ORAL COMMUNICATIONS S1: Supramolecular Assemblies

Transcription

XIV Congress of the Spanish Biophysical Society (SBE 2014)
ORAL COMMUNICATIONS
S1: Supramolecular Assemblies
S1.OP1. Functional nanostructures by designed protein self-assembly. (P07)
Sara Hernández Mejías, Pierre Couleaud, Javier López, Begoña Sot, Carmen Atienza,
Teresa González and Aitziber L. Cortajarena.
S1.OP2. A common link for programmed cell death in humans and plants. (P14)
Irene Díaz-Moreno, Jonathan Martínez-Fábregas, Katiuska González-Arzola, Antonio
Díaz-Quintana and Miguel A. De la Rosa.
S1.OP3. TubZ filaments conformational change underlying GTP hydrolysis: c-terminal
tail implication in the opening of the protofilament twist. (P16)
Maria A. Oliva Blanco, María Eugenia Fuentes and Fernando Moreno.
S1.OP4. Replication initiation proteins studied with AFM. (P35)
María Eugenia Fuentes-Pérez, Katarzyna Wegrzyn, Igor Konieczny and Fernando
Moreno-Herrero.
---------
S1-2.OP5. ITC and DSC techniques for studies of biological macromolecules.
Peter Vikegard.
FUNCTIONAL NANOSTRUCTURES BY DESIGNED PROTEIN SELF-ASSEMBLY
Sara H. Mejías1,2, Pierre Couleaud1,2, Javier López1,3, Begoña Sot1,2, Carmen Atienza1,3,
Teresa González1, Aitziber L. Cortajarena1,2
1
Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia)
28049-Madrid, Spain.
sara.hernandez@imdea.org
2
CNB-CSIC-IMDEA Nanociencia Associated Unit "Unidad de Nanobiotecnología"
3
Departamento de Química Orgánica, Facultad de C.C. Químicas, UCM, Madrid, Spain.
The development of application-oriented innovative materials requires methods for control of
structures along different size scales. Bottom-up self-assembly that relies on highly specific
biomolecular interactions of small defined components, is an attractive approach for biomaterial
design and nanostructure templating1.
In this work, we used modular designed consensus tetratricopeptide repeat proteins (CTPRs)
(Figura1A)2 for the generation of 3D structures (protein nanotubes) and functional nanostructures.
CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface
to form elongated superhelix (Figure 1B)3,4.
We present the design and characterization of 3D protein-structures to use them as templates for
the creation of functional materials. We show the conjugation and characterization of the protein
with gold nanoparticles (Au-Nps) and preliminary results of the conjugation of proteins with
electroactive organic molecules.
1. The formation of the nanotubes is achieved by introducing two single point mutations in the
protein to create a new hydrophobic interface in the CTPR superhelix that will interact with
identical interface on another CTPR molecule (Figure 1C). We show the formation of protein
nanotubes and characterization of the new 3D structures.
2. The conjugation of gold nanoparticles to CTPR protein templates aims for the organization of
nanometric objects with atomic precision. Specific amino acids for metal coordination and
nanoparticle binding are introduced at defined positions of the CTPR repeat sequence. We use
high resolution imaging techniques such as atomic force microscopy (AFM), transmission
electron microscopy (TEM) and scanning transmission microscopy (STM) to characterize the
CTPR-AuNps conjugates. (Figure1D).
3. We propose to use CTPR proteins in order to template donor-acceptor pairs for electroactive
materials. In order to achieve an efficient electron transfer the arrays of molecules need to be
ordered with defined inter-molecular distances. We show preliminary conjugation data in which
the potential of CTPR protein scaffolds for nanometer-precise arrangement of the molecules is
explored (Figure 1E).
References
1) T. Z. Grove, L. Regan and A. L. Cortajarena,
J. R. Soc. Interface., 2013, 10, 20130051.
(2) Kajander, T.; Cortajarena, A. L.; Regan, L.
Methods Mol. Biol. 2006, 340, 151.
(3) Kajander, T.; Cortajarena, A. L.; Mochrie, S.
G.; Regan, L. Act Crystallographica 2007,
D63, 800.
(4) Cortajarena, A. L.; Wang, J.; Regan, L. Febs
J 2010, 277, 1058.
A COMMON LINK FOR PROGRAMMED CELL DEATH
IN HUMANS AND PLANTS
Irene Díaz-Moreno, Jonathan Martínez-Fábregas, Katiuska González-Arzola,
Antonio Díaz-Quintana and Miguel A. De la Rosa
Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartuja, Universidad de Sevilla – CSIC, Sevilla, Spain
idiazmoreno@us.es
Programmed cell death (PCD) is a fundamental event for the development of multicellular
organisms. In mammalian cells, early events in PCD involve the release of cytochrome c
(Cc) from mitochondria to the cytoplasm to act at the first stages of the apoptotic process,
playing a key role in assembling the apoptosome. In plants, PCD is part of a general
process named hypersensitive response, where Cc is also released into the cytosol but its
role in PCD remains veiled. Such highly conserved cytoplasmic location of Cc upon
apoptotic stimuli lead to think of a common link for PCD in evolutionarily distant species,
like humans and plants.
To better understand the role of Cc in the onset of PCD in both humans and plants, a
proteomic approach based on affinity chromatography with Cc as bait was used. Upon
combining this approach and Bimolecular Fluorescence Complementation (BIFC), a total
of 8 human and 9 plant new proteins interacting with Cc under PCD were found [1,2].
These new PCD Cc-partners are involved in protein folding, translational regulation,
oxidative stress, DNA damage, energetic and mRNA metabolism. Strikingly, some of the
novel human Cc-targets are closely related to those for plant Cc, indicating that the
evolutionarily well-conserved cytosolic Cc – appearing in organism from plant to
mammals – interact with a wide range of targets on PCD.
Modeling of the complexes between human and plant Cc with its counterparts shows how
the heme crevice of Cc takes part of the complex interface in agreement with the vast
majority of known redox adducts of Cc. However, in contrast to the high turnover rate of
the mitochondrial Cc redox adducts, those occurring under PCD lead to the formation of
rather stable nucleo-cytoplasmic ensembles, as inferred from Surface Plasmon Resonance
(SPR) and Nuclear Magnetic Resonance (NMR) measurements. On the basis of these
findings, we suggest that human and plant Cc interacts with pro-survival, anti-apoptotic
proteins after its release into the cytoplasm. Then, Cc may interfere with cell survival
pathways and unlock PCD in order to prevent the spatial and temporal co-existence of
antagonist signals.
Acknowledgements: This work has been sponsored by the Spanish Ministry of Economy and
Competitiveness (BFU2012-31670) and the Regional Government of Andalusia (BIO198).
References
[1] Martínez-Fábregas J, Díaz-Moreno I, González-Arzola K, Janocha S, Navarro JA, Hervás M,
Bernhardt R, Díaz-Quintana A, De la Rosa MA, “New Arabidopsis thaliana cytochrome c partners:
a look into the elusive role of cytochrome c in programmed cell death in plants”, Mol. Cell.
Proteomics, 12 (12), 3666-3676, 2013.
Bernhardt R, Velázquez-Campoy A, Díaz-Quintana A, De la Rosa MA, “Structural and functional
analysis of novel human cytochrome c targets in apoptosis”, Mol. Cell. Proteomics,
doi:10.1074/mcp.M113.034322, 2014.
TubZ FILAMENTS CONFORMATIONAL CHANGE UNDERLYING
GTP HYDROLYSIS: C TERMINAL TAIL IMPLICATION
IN THE OPENING OF THE PROTOFILAMENT TWIST
María A. Oliva1, María E. Fuentes2 and Fernando Moreno2
1
CSIC-Centro de Investigaciones Biológicas. c/ Ramiro de Maeztu, 9. 28040-Madrid, Spain.
marian@cib.csic.es
2
CSIC- Centro Nacional de Biotecnología. c/ Darwin, 3. 28049-Madrid, Spain
TubZ is the latest described member in tubulin/FtsZ superfamily of self-assembly GTPases and
is involved in plasmids and phages DNA distribution within the bacteria [1, 2, 3]. Showing a
tubulin-like polar protofilament formation, TubZ assembles into double helical filaments [4] that
self-organize and follow a treadmilling dynamics, similarly to actin filaments [5, 6].
Considering that dynamicity is crucial for the function of cytomotive filaments, we have studied
the structural conformational change in TubZ filaments due to GTP hydrolysis. We have used a
biochemical and structural approach to understand the direction of the changes and how they drive
the disassembly process. We have found that the transition is different from the straight-to curved
change described in other tubulin-like filaments. Instead, in TubZ filament there is an opening of
the helical structure by increasing of the inter-monomer twist (straightening of the protofilament),
which becomes incompatible with treadmilling dynamics. Further, the C-terminal tail is: i)
responsible of the formation of a functional double helical filament by getting the appropriate
inter-monomer twist, ii) necessary for spreading the changes induced by GTP hydrolysis along
the filaments and, iii) important in the interaction with its partner protein TubY during the remodeling of the filaments into FtsZ/tubulin-like rings structures.
Acknowledgements: This work has been sponsored by RyC program (2011) and Jose M. Andreu’s
grant BFU2011-23416.
References
[1] L Ni, W Xu, M Kumaraswami, MA Schumacher, Plasmid protein TubR uses a distinct mode of HTHDNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition, Proc Natl
Acad Sci U S A, 107 (26), 11763-8, 2010.
[2] MA Oliva, AJ Martin-Galiano, Y Sakaguchi, JM Andreu, Tubulin homolog RubZ in a phage-encoded
partition system, Proc Natl Acad Sci U S A, 109 (20), 7711-6, 2012.
[3] JA Kraemer, ML Erb, CA Waddling, EA Montabana, EA Zher, H Wang, K Nguyen, DS Pham, DA
Agard, J Pogliano, A phage tubulin assembles dynamic filaments by an atypical mechanism to center
viral DNA within the host cell, Cell, 149 (7), 1488-99, 2012.
[4] CH Aylett, T Izore, LA Amos, J Löwe, Filament structure of bacterial tubulin homologue TubZ, Proc
Natl Acad Sci U S A, 107 (46), 19766-71, 2010.
[5] Y Chen, HP Erickson, In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus
plasmids: evidence for a capping mechanism, J Biol Chem, 283 (13), 8102-9, 2008.
[6] RA Larsen, C Cusumano, A Fujioka, G Lim-Fong, P Patterson, J Pogliano, Treadmilling of a
prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis, Genes
Dev, 21 (11), 1340-52, 2006.
REPLICATION INITIATION PROTEINS STUDIED WITH AFM
Maria Eugenia Fuentes-Perez1, Katarzyna Wegrzyn2, Igor Konieczny2, and
Fernando Moreno-Herrero1
1
Centro Nacional de Biotecnología, CSIC, Cantoblanco, Madrid, Spain
me.fuentes@cnb.csic.es
2
University of Gdansk, Gdansk, Poland.
DNA replication is a fundamental cellular process whose mechanism is still not well understood.
Replication requires a specific DNA region, known as the origin of replication (Ori), as well as
specific proteins, called replication initiation proteins (Rep). Both DNA and proteins form the
replication initiation complex. The origin of replication in plasmids and phage DNA contains
some conserved elements. These include specific binding sites (iterons) for Rep proteins, DnaA
boxes for DnaA proteins and an AT-rich region where DNA melting occurs. In this work, we used
the Atomic Force Microscope (AFM) to study the binding of Rep proteins to the origin of
replication in the broad-host-range plasmid RK2 [1]. The origin of replication in RK2 plasmid is
called OriV. It possesses 5 iterons where the replication initiation protein TrfA binds, four DnaA
boxes for DnaA proteins and four 13-meres in the AT rich region [2]. Using the AFM, we were
able to capture the binding of TrfA to the iterons region. Interestingly, while bound to the iterons,
TrfA also interacts with a ssDNA oligonucleotide containing the sequence of one of the strands
of the AT rich region. Moreover, the TrfA-ssDNA interaction is dependent on the sequence of
the oligonucleotide. Our AFM approach was also applied to RepE protein, a replication initiation
protein from plasmid F. Notably, we found that binding of RepE was also favored by the
equivalent ssDNA oligonucleotide of the AT-rich region of plasmid F. These findings enable to
create a general model in which firstly, Rep proteins induces the melting of the AT-rich region
and secondly, specific interaction of Rep protein with one of the melted ssDNA occur.
Acknowledgements: This work has been supported by a Starting Grant from the European Research
Council [grant number 206117] (MEFP and FMH) and by a grant from the Spanish Ministry of Science
and Innovation [grant number FIS2011-24638] (FMH). The work in Poland was supported by the Polish
National Science Centre [grant number 24 2012/04/A/NZ1/00048] (KW and IK).
References
[1] Doran, K.S., I. Konieczny, and D.R. Helinski, Replication Origin of the Broad Host Range Plasmid
RK2. Journal of Biological Chemistry, 273(14): p. 8447-8453, 1998.
[2] Rajewska, M., K. Wegrzyn, and I. Konieczny, AT-rich region and repeated sequences–the essential
elements of replication origins of bacterial replicons. FEMS microbiology reviews, 36(2): p. 408434, 2011.
ITC AND DSC TECHNIQUES FOR STUDIES
OF BIOLOGICAL MACROMOLECULES
Dr. Peter Vikegard
TA Instruments
The term microcalorimetry was coined a couple of decades ago, with the prefix microreferring to the sensitivity of the technique in terms of the measured thermal power, i.e.
microwatts or micro calories per unit of time. The technological advances of calorimetry
since then have been such that we can today talk about nanowatts rather than microwatts
suggesting that nanocalorimetry might be a more appropriate term.
This advancement of the technology has, at least partially, been driven by the necessity
of working with very small sample masses, especially regarding proteins. Large effort has
to be put into the production and purification of small amounts proteins with large costs
as a consequence.
With the ITC (Isothermal Titration Calorimetry) technology processes are initiated in a
calorimeter by injecting and mixing one component with another. Most commonly these
processes are physical interaction between the components such as protein-protein,
protein-small drug, protein-lipid etc. The information gained is a complete
thermodynamic profile of the interaction, in terms of the binding affinity and the free
energy-, enthalpy- and entropy change for the process. ITC can also shed light on
stoichiometry and mechanism of binding, e.g. competitive, several sets of binding sites
etc. In addition to this ITC has been used as a kinetic tool for slower processes such as
enzyme catalyzed reactions. A chemical reaction is initiated by injecting a substrate into
an enzyme solution, whereby the heat flow reflects the rate of the process. This enables
determination of appropriate kinetic model as well as rate constants and catalytic constant
to be determined.
Hitherto the binding affinity for intermolecular interactions obtained from ITC has been
the main interest for researchers working with this technique. However, in recent years
attention has been drawn to “thermodynamic profiling” in the design of new drugs as this
provides a deeper understanding of the driving forces responsible for molecular
interactions. The question that is addressed in this respect is to what extent a binding is
driven by the changes in entropy and enthalpy. Modelling of the ITC results is normally
performed with the binding affinity and the enthalpy change as a set of independent
parameters while the entropy change is obtained as a dependent parameter through Gibbs
equation. Hence in order to get reliable determination of the thermodynamic profile
accurate determination of the enthalpy change for the binding is important. The
measurement of the heat generated or absorbed by a process is non-specific. Besides the
heat effects of binding, heat of dilution, heat of injection, non-specific interaction, buffer
effects etc. may be significant to the total measured heat. It is thus important to correct
for these effects by designing proper control experiments.
In this presentation, the contributors to the overall heat generated in a typical ITC
experiment will be discussed as well as to what extent we can correct for the events that
is not included in the molecular recognition process.
ORAL COMMUNICATIONS
S2: Receptors, Channels and Transporters
S2.OP1. Physiological role of the collaboration of Kv1.3 with caveolin during
adipogenesis. (P23)
Mireia Pérez-Verdaguer, Jesusa Capera, Clara Serrano-Novillo, Joanna Bielańska,
Marta Camps, Anna Gumà, Núria Comes and Antonio Felipe.
S2. OP2. The Transmembrane domain interaction network of Bcl-2 family members. (P48)
Vicente Andreu Fernández, Ainhoa Genovés, Ismael Mingarro, Mar Orzáez and
Enrique Pérez Payá.
S2.OP3. Kv7.1/Kv7.5 heterotetramers with emerging properties on vascular smooth
muscle physiology. (P29)
Anna Oliveras, Meritxell Roura-Ferrer, Laura Solé, Alicia de la Cruz, Ángela Prieto,
Ainhoa Etxebarria, Joan Manils, Daniel Morales-Cano, Enric Condom, Concepció Soler,
Angel Cogolludo, Carmen Valenzuela, Alvaro Villarroel, Núria Comes and Antonio Felipe.
S2.OP4. The S6-TRPbox linker domain in TRPM8 plays a central role in allosteric
channel activation. (P62)
Francisco José Taberner, Ainara López-Córdoba, Gregorio Fernández-Ballester, Yuri
Korchev and Antonio Ferrer-Montiel.
PHYSIOLOGICAL ROLE OF THE COLLABORATION OF Kv1.3
WITH CAVEOLIN DURING ADIPOGENESIS
Mireia Pérez-Verdaguer1,2, Jesusa Capera1,2, Clara Serrano-Novillo1,2, Joanna Bielańska1,2,
Marta Camps2, Anna Gumà2, Núria Comes1,2 and Antonio Felipe1,2
1
Molecular Physiology Laboratory, 2Departament de Bioquímica i Biologia Molecular,
Institut de Biomedicina (IBUB), Universitat de Barcelona. (Spain)
The voltage-dependent K+ channel Kv1.3 is involved in a myriad of physiological events
in leukocytes, sensory neurons, vascular smooth muscle and adipocytes. Kv1.3
concentrates in lipid rafts. These domains are signal platforms where signalling molecules
and targets converge. Specialized forms of rafts are caveolae. These omega shaped
structures are highly abundant in adipocytes where they account for 30% of the plasma
membrane surface. Their structure is due to the presence of caveolin 1 which participates
in the transport of cholesterol from endoplasmic reticulum to plasma membrane.
The role of Kv1.3 in adipocytes raises an important debate as it has been proposed that
Kv1.3 could be a pharmacological target in obesity. Because the localization of the
channel is important for its function, in the present work we studied the presence and
localization of Kv1.3 in adipocytes. We have characterized the presence of Kv1.3 in rat
and human adipocytes and during adipogenesis of the 3T3-L1 cell line. Adipogenesis
leads to caveolin 1 (Cav1) expression and the appearance of caveolae; therefore, we
analyzed the microdomian localization of Kv1.3. In addition, by lentiviral infection, we
generated different 3T3-L1 cell lines genetically deficient in caveolin 1 and Kv1.3, in
order to study any putative phenotypically reprograming. We found that in Cav1-null
cells, Kv1.3 shifted floatability. In addition, Kv1.3-null cells did not reach adipocyte
differentiation because an impaired proliferative behaviour. Our results indicate that
adipogenesis triggers a relocalization of Kv1.3 in newly synthesized caveolae. Further,
Kv1.3 is crucial for the regular cell cycle progression in 3T3-L1 pre-adipocytes. Because
most of insulin-dependent adipocyte signalling machinery is located in these structures,
our results bring light to the role of Kv1.3 in the adipocyte physiology.
Supported by BFU2011-23268 and CSD2008-00005 from the MINECO, Spain
THE TRANSMEMBRANE DOMAIN INTERACTION NETWORK
OF Bcl-2 FAMILY MEMBERS
Vicente Andreu-Fernández1, Ainhoa Genovés1, Ismael Mingarro2 , Mar Orzáez1 and
Enrique Pérez Payá
1
Centro de Investigación Príncipe Felipe, Valencia, España;
vandreu@cipf.es
2
Universidad de Valencia, Valencia, España.
Intrinsic apoptosis in mammals is regulated by protein-protein interactions among the Bcell lymphoma-2 (Bcl-2) family1. The relevance of Bcl’s BH3 cytosolic domain in
defining the protein-protein interactions between different pro- and anti- apoptotic
members has been extensively analyzed2. However, the function of the transmembrane
domain (TMD) in such interactions has not been yet defined. It is becoming apparent that
the TMDs are more than mere insertion domains and may play a key role in the function
of the Bcl-2 proteins3. We have analyzed the contribution of TMDs to the regulation of
protein interactions among the different pro- and anti-apoptotic Bcl-2 members and their
relevance to the control of the apoptotic cellular fate. To accomplish these objectives we
have performed an overall study of the TMD protein-protein interactions among the
different Bcl-2 proteins4,5. The results of this study have prompted us to define the Bcl-2
TMD interaction map.
Acknowledgements: This work has been sponsored by grants from the Spanish Ministry of Science and
Innovation (MICINN-BIO2007-60066, -SAF2010 15512, -SAF2008-00048 and CSD2008-00005C), and
by Generalitat Valenciana (GV) Prometeo 2010/005.
References
1. Llambi F, Moldoveanu T, Tait SW, Bouchier-Hayes L, Temirov J, McCormick LL, Dillon CP,
Green DR. A unified model of mammalian BCL-2 protein family interactions at the mitochondria.
Mol Cell; 44(4):517-31. (2011).
2. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. The BCL-2 family reunion. Mol
Cell; 37(3):299-310. (2010).
3. Lindsay J, Esposti MD, Gilmore AP. Bcl-2 proteins and mitochondria Specicity in membrane
targeting for death. Biochimica et Biophysica Acta 1813, 532–539 (2011).
4. Yin H, Slusky JS, Berger BW, Walters RS, Vilaire G, Litvinov RI, Lear JD, Caputo GA, Bennett
JS, DeGrado WF. Computational Design of Peptides That Target Transmembrane Helices.
Science; 315(5820):1817-22 (2007).
5. Zhu H, Metcalf DG, Streu CN, Billings PC, Degrado WF, Bennett JS. Specificity for
homooligomer versus heterooligomer formation in integrin transmembrane helices. J Mol Biol.
401(5):882-91 (2010).
Kv7.1/Kv7.5 HETEROTETRAMERS WITH EMERGING PROPERTIES
ON VASCULAR SMOOTH MUSCLE PHYSIOLOGY
Anna Oliveras1, Meritxell Roura-Ferrer1,2, Laura Solé1, Alicia de la Cruz3, Angela Prieto3,
Ainhoa Etxebarria2, Joan Manils4, Daniel Morales-Cano5, Enric Condom4,
Concepció Soler4, Angel Cogolludo5, Carmen Valenzuela3, Alvaro Villarroel2,
Núria Comes1, Antonio Felipe1.
1
Molecular Physiology Laboratory, Departament de Bioquímica i Biologia Molecular,
Institut de Biomedicina (IBUB), Universitat de Barcelona.
2
Unidad de Biofísica, CSIC-UPV/EHU, Universidad del País Vasco.
3
Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-Universidad Autónoma de Madrid.
4
Departament de Patologia i Terapèutica Experimental, Hospital Universitari de BellvitgeUniversitat de Barcelona.
5
Departamento de Farmacología, Universidad Complutense de Madrid,
Ciber Enfermedades Respiratorias (CibeRes), Spain
Voltage-dependent K+ channels from Kv7 (KCNQ) family have well-established
physiological roles in cardiovascular and nervous system, although functions in
blood vessels remain unclear. Kv7.1, Kv7.4 and Kv7.5 are predominant in vascular
smooth muscle. Evidence suggests a role controlling vascular reactivity in several
smooth muscles. However, because controversial pharmacological results Kv7.1 is
under intense investigation. Therefore, establishing the entities that control smooth
muscle tone is a topic of interest. In this scenario, the ability of Kv7 channels to form
heterotetramers is of physiological relevance. Thus, the confirmation of Kv7.4/Kv7.5
heterotetramers paves the way for novel interaction that could shed light to
controversial pharmacological results. We aim whether Kv7.1 and Kv7.5 may form
heterotetrameric channels that could increase diversity on channel response in
vascular smooth muscle cells. We proved the presence of Kv7.1/Kv7.5 structures in
heterologous system by many different approaches, such as electrophysiology, coimmunoprecipitation and FRET experiments. Heteromeric channels are mainly
retained at the endoplasmatic reticulum and, unlike homomeric Kv7.1 channels,
heteromers localize out of lipid raft microdomains. These results are supported by
experiments in isolated smooth muscle myocytes. We demonstrated that Kv7.1 and
Kv7.5 are differentially expressed in aorta, cava and coronary myocytes.
Electrophysiological and miography recordings using linopiridine, chromanol 293B
and retigabine suggested that Kv7.1/Kv7.5 form heterotetramers. Coimmunoprecipitation experiments further confirmed the hypothesis. Finally, lipid raft
isolation from different tissues corroborated that predominant expression of Kv7.5
releases Kv7.1/Kv7.5 oligomers out of lipid raft structures. Our findings demonstrate
that Kv7.1 and Kv7.5 are differentially expressed in several types of blood vessels
where they contribute to control vascular reactivity. We prove that they do
heterotetramerize increasing the diversity of their physiological response. These data
may help to better understand the scenario of Kv7 channels and vascular physiology.
Supported by BFU2011-23268 and CSD2008-00005 to AF (MINECO, Spain)
THE S6-TRPBOX LINKER DOMAIN IN TRPM8 PLAYS A CENTRAL ROLE
IN ALLOSTERIC CHANNEL ACTIVATION
Francisco José Taberner1, Ainara López-Córdoba1,2, Gregorio Fernández-Ballester1,
Yuri Korchev2 and Antonio Ferrer-Montiel1,3
1
Instituto de Biología Molecular y Celular. Universidad Miguel Hernández. Elche. Spain.
2
Imperial Collage School of Medicine, London, United Kingdom.
3
BIOFISIKA, the Basque Center for Biophysics, UPV/EHU-CSIC-FBB, Bilbao. Spain.
The ability of TRP channels to sense and respond to environmental and endogenous cues
is crucial in animal sensory physiology. However, the molecular mechanism of channel
gating is not well understood. The cytosolic C-terminus domain is considered to be
strategic in the channel opening. Recently, it has been pointed out that TRP box in the Cterminus is pivotal for allosteric activation in this channel family. Here, we have
examined the role of the linker region between the TRPM8 inner gate and the TRP box
(referred to as the S6-TRPbox linker) to identify structural determinants of channel
gating. Using Step-wise substitutions of segments in the S6-TRPbox linker of TRPM8
channel with the cognate TRPV1 sequences, we identified Y981 and V986 as central
molecular determinants of channel function. Notably, mutation of these two positions in
a chimeric and wild type channels had a great impact on channel gating by voltage and
menthol, as evidenced by the modulation of the conductance-to-voltage (G-V)
relationships. Simulation of G-V curves using an allosteric model for channel activation
revealed that these mutations altered the allosteric constants that couple stimuli sensing
to pore opening. A molecular model of TRPM8, based on the recently reported TRPV1
structural model, showed that Y981 lies in a hydrophobic pocket at the end of the S6
transmembrane segment and is involved in inter-subunit interactions with residues from
neighbour subunits. V986 appears located in a tight hydrophobic cavity making intrasubunit interactions with residues of the S4-S5 linker. These findings substantiate a gating
mechanism whereby the TRP domain acts as a coupling domain for efficient channel
gating. Furthermore, they imply that protein-protein interactions of the TRP domain may
be targets for channel modulation and drug intervention.
ORAL COMMUNICATIONS
S4: Protein Folding, Stability, Function and Dynamics
S4.OP1. Venezuelan equine encephalitis virus nsP1: mechanism of action of a viral
capping enzyme. (P13)
Jaime Guillén, Changqing Li, Julie Lichière, Bruno Canard, Etienne Decroly and Bruno
Coutard.
S4.OP2. Mechanical stability of the natural DnaJ Zn-finger domain revealed by single
molecule force spectroscopy. (P17)
Judit Perales-Calvo, Ainhoa Lezamiz and Sergi García-Manyes.
S4.OP3. Apocynin inhibits purified cytochrome b5 reductase activity by competition
with NADH for its active site on the protein. (P66)
Alejandro K. Samhan Arias and Carlos Gutiérrez-Merino.
S4.OP4. FIREBALL/AMBER: QM/MM Method for biomolecular systems. (P78)
Jesús I. Mendieta Moreno, Ross C. Walker, James P. Lewis, Paulino Gómez-Puertas,
Jesús Mendieta and José Ortega.
VENEZUELAN EQUINE ENCEPHALITIS VIRUS NSP1:
MECHANISM OF ACTION OF A VIRAL CAPPING ENZYME
Jaime Guillén, Changqing Li, Julie Lichière, Bruno Canard,
Etienne Decroly and Bruno Coutard
CNRS and Aix Marseille Université, UMR7257,
Architecture et Fonction des Macromolécules Biologiques, Marseille, France
j_guillen1980@hotmail.com
Venezuelan equine encephalitis virus (VEEV) is an infectious pathogen belonging to the
New World alphaviruses. Many of these alphaviruses are important human pathogens.
Their genomic and sub-genomic RNA are protected by a 5’ end cap structure, which is
essential for the translation of viral proteins. The nonstructural protein 1 (nsP1) is the
main enzyme for RNA capping in alphavirus. nsP1 catalyses the methylation at position
N7 of a GTP molecule and then forms a covalent link with the N7 methylated GMP,
releasing PPi. It is then supposed that this N7 Methyl GMP is transferred on the viral
mRNA. Both Methyltransferase (MTase) and Guanylyltransferase (GTase) activities of
VEEV nsP1 were characterized by means of original assays in order to uncouple both
reactions. Critical amino acids positions for both MTase and GTase functions were next
identified by site directed mutagenesis. In parallel, the binding of various RNAs and NTP
on nsP1 were characterized by biophysical methods to strengthen biochemical data.
Finally, these methods were used to evaluate the inhibition of guanylyltransferase activity
by small molecules.
Acknowledgements: This work has been sponsored by IEF Marie Curie fellowship, EUVIRNA (Marie
Curie Initial Training Network) and by the project SILVER (Health-F3-2010-260644) of the
European Union 7th Framework Program.
MECHANICAL STABILITY OF THE NATURAL DnaJ ZN-FINGER DOMAIN
REVEALED BY SINGLE MOLECULE FORCE SPECTROSCOPY
Judit Perales-Calvo, Ainhoa Lezamiz and Sergi Garcia-Manyes
Department of Physics and Randall Division of Cell and Molecular Biophysics,
King’s College London, Strand, London WC2R 2LS
Zinc is an abundant metal essential for life that is present in ca.2800 proteins in the human
proteome. When coordinated inside a protein structure it often forms a stable structural
motif, the so-called zinc finger [1]. DnaJ is a 5-domain Hsp40 molecular chaperone that
naturally binds two atoms of Zn2+, each one coordinated to two –CXXC- chelating motifs,
acquiring an unusual tetrahedral C4-type Zn-finger topology [2]. Here we use single
molecule force spectroscopy AFM to unravel the mechanical stability of each
independent DnaJ zinc finger. Upon pulling the protein at a constant velocity, we first
discover that a 3-domain mutant of DnaJ (DnaJ107) follows a sequential unfolding
mechanism that does not follow a common mechanical hierarchy scenario. On a typical
unfolding trajectory, the mechanically labile domain III unfolds first, requiring a force of
105  44 pN, followed by the unfolding of domain I, occurring at 191  39 pN. Crucially,
the hidden Zn binding domain is exposed to the mechanical force only when domain I
has already unfolded. The force required to break an individual Zn-S bond, triggering the
unfolding of both Zn fingers, is surprisingly low, of 90  11pN. Pulling on an Apo DnaJ
mutant (DnaJ107(C161/164/197/200S)), whereby two cysteines are mutated in each finger to
impair Zn2+ binding, reveals that in half of the trajectories the apo-form (devoid of
mechanical stability) is formed. However, in the other half of the trajectories, a single
new ZnS4 centre that features a mechanical stability akin to that of both native fingers is
unexpectedly observed, demonstrating the plasticity of the structure as both Zn-fingers in
DnaJ are able to hybridize. Our results provide direct quantification of the mechanical
role of individual Zn-thiolate bonds, of widespread presence in nature.
[1] Berg, JM. and Shi, Y. (1996). Science. 271: 1081-5.
[2] Martinez-Yamout, M., Legge, GB., Zhang, O., Wright, PE. and Dyson, HJ. (2000). J Mol Biol. 300:
805-18.
APOCYNIN INHIBITS PURIFIED CYTOCHROME B5 REDUCTASE
ACTIVITY BY COMPETITION WITH NADH FOR ITS ACTIVE SITE
ON THE PROTEIN
Alejandro K. Samhan Arias1 and Carlos Gutiérrez-Merino
Dept. Biochemistry and Molecular Biology, Faculty of Sciences,
University of Extremadura, 06006-Badajoz, Spain
aksamhan@unex.es
Cytochrome b5 reductase (Cb5R) is a pleiotropic flavoprotein that catalyzes multiple one
electron reduction reactions in cells. In previous works, we have shown that Cb5R can
account for most of NADH dependent superoxide anion production of the neuronal
plasma membrane, being largely responsible of the superoxide anion overshot that is
observed during apoptosis of cerebellar granule neurons induced by extracellular
potassium deprivation [1]. In addition, NADPH oxidases have also been shown to be
sources of O2·- at the plasma membrane of cells under apoptotic stimulus in other type of
neurons [2]. Apocynin is one of the most frequently used inhibitors of NOX activity
inhibiting the plasma membrane NADPH oxidase activity of different cell types. We
found that 1mM of apocynin, a standard concentration used in culture to block NOX
activity, strongly inhibits the O2·- production by purified Cb5R, e.g. 79.7±13.7%
inhibition. Moreover, apocynin was also found to be a potent inhibitor of the NADH:
ferricyanide reductase activity of purified Cb5R, displaying an inhibition constant of 75
μM and reaching 90.1±10.4% inhibition with only 200 μM of apocynin. The analysis of
docking simulations suggested that apocynin inhibition of Cb5R activities is produced by
binding of this compound to the protein domain where the NADH binding site is located.
This hypothesis was experimentally assessed by kinetic analysis.
Acknowledgements
This work has been supported by Grant BFU2011-30178 of the Spanish Plan Nacional de I+D+I and by
Grant GR10092 of the Gobierno de Extremadura with FEDER co-financing.
References
[1] Samhan-Arias, A. K., Garcia-Bereguiain, M. A., Martin-Romero, F. J. and Gutierrez-Merino, C.
Clustering of plasma membrane-bound cytochrome b5 reductase within 'lipid raft' microdomains of
the neuronal plasma membrane. Mol. Cell. Neurosci. 40: 14-26; 2009.
[2] Sorce, S. and Krause, K. H. NOX enzymes in the central nervous system: from signaling to disease.
Antioxid. Redox Signal. 11: 2481-2504; 2009.
FIREBALL/AMBER: A QM/MM METHOD FOR BIOMOLECULAR SYSTEMS
Jesús I. Mendieta-Moreno1,2,3, Ross C. Walker4, James P. Lewis5,
Paulino Gómez-Puertas2, Jesús Mendieta2,3 and José Ortega1
1
Departamento de Física Teórica de la Materia Condensada and Condensed Matter Physics Center
(IFIMAC), Universidad Autónoma de Madrid, ES-28049 Madrid, Spain
2
Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), ES-28049 Madrid, Spain
3
Biomol-Informatics SL, Campus UAM, ES-28049 Madrid, Spain
4
San Diego Supercomputer Center and Department of Chemistry and Biochemistry,
University of California-San Diego, La Jolla, California 92093, United States
5
West Virginia University, Morgantown, West Virginia 26506-6315, United States
In recent years, quantum mechanics/molecular mechanics (QM/MM) methods have
become an important computational tool for the study of chemical reactions and other
processes in biomolecular systems. Because of the complexity of biomolecules and the
desire to achieve converged sampling, it is important that the QM method presents a good
balance between accuracy and computational efficiency. Here, we report on the
implementation of a QM/MM technique that combines a DFT approach specially
designed for the study of complex systems using first-principles molecular dynamics
simulations (FIREBALL) with the AMBER force fields and simulation programs [1].
We also present the application of this method, using DFT QM/MM molecular dynamics
techniques, to study two different enzymatic reactions: phosphodiester bond cleavage by
RNase A and DNA polymerization by HIV reverse transcriptase. In particular, the
computational efficiency of our approach allowed the generation of free-energy surface
maps to explore the large conformational space for the reactions, thus permitting a
detailed analysis of alternative pathways.
Reference
[1] Jesús I. Mendieta-Moreno, Ross C. Walker, Paulino Gómez-Puertas, James P. Lewis, Jesús Mendieta
and José Ortega, “FIREBALL/AMBER: An efficient local-orbital DFT QM/MM method for
biomolecular systems”, Journal of Chemical Theory and Computation, 10 (5), 2185–2193, 2014.
ORAL COMMUNICATIONS
S5: Protein Structure
S5.OP1. Conformational selection mechanism in protein-protein association: insights
from docking. (P04)
Chiara Pallara, Manuel Rueda and Juan Fernández-Recio.
S5.OP2. Stabilization of the integrin β4-plectin interaction with thiol reactive
fluorescent compounds. (P82)
José M. de Pereda, José A. Manso and Stephane Boivin.
S5.OP3. How allosteric control of Staphylococcus aureus penicillin-binding protein 2a
enables methicillin-resistance and physiological function. (P30)
Juan Antonio Hermoso, Lisandro H. Otero, Alzoray Rojas-Altuve, Leticia I. Llarrull, César
Carrasco-López, Malika Kumarasiri, Elena Lastochkin, Jennifer Fishovitz, Matthew
Dawley, Dusan Hesek, Mijoon Lee, Jarrod W. Johnson, Jed F. Fisher, Mayland Chang
and Shahriar Mobashery.
S5.OP4. Structural basis for the modulation of tubulin by antitumoral drugs. (P49)
José Fernando Díaz Pereira, Andrea Prota and Michel O. Steinmetz.
CONFORMATIONAL SELECTION MECHANISM IN
PROTEIN-PROTEIN ASSOCIATION: INSIGHTS FROM DOCKING
Chiara Pallara, Manuel Rueda and Juan Fernández-Recio
Joint BSC-IRB Research Programme in Computational Biology, Barcelona Supercomputing Center,
Barcelona, Spain . E-Mail: chiara.pallara@bsc.es
To understand cellular processes at molecular level we need to improve our knowledge
of protein-protein interactions, but determining the atomic structure of many protein
complexes is still challenging. Thus, structural prediction of protein-protein association
is one of the major goals of computational biophysics. Despite methodological advances
in docking protocols, dealing with molecular flexibility is a major bottle-neck, as the
experiment CAPRI (Critical Assessment of PRediction of Interactions) [1] has shown.
Indeed, state-of-the-art rigid-body docking approaches like pyDock [2] show excellent
success rates [3], but have difficulties in cases with large conformational changes upon
binding [4]. For complexes that form via conformational selection mechanism, in which
the unbound state can sample bound conformers, a largely unexplored strategy to include
flexibility in docking predictions would consist on the use of precomputed
conformational ensembles generated from unbound protein structures [5]. Recently we
applied this approach to a series of ubiquitin complexes, in which the use of RDCderived
ensembles significantly improved docking predictions [6]. Here, we have extended this
strategy to the set of 124 cases in Protein-Protein Docking Benchmark 3.0 [7].
Conformational ensembles for the unbound docking partners were automatically
generated by using three different computational approaches, modeling minimization
(MM), molecular dynamics (MD) and normal mode analysis (NMA). To establish the
limits of the approach in optimal conditions, we first used for docking only those
conformers that would be expected to give best results based on their similarity to the
bound structure. Then, for a small sub-set of cases we devised a more realistic protocol
by using all conformers for the docking simulations. The results show that the use of small
conformational ensembles can significantly improve docking predictions in high-affinity,
medium-flexibility complexes. In addition to the relevance for methodology
development, his work shows that the definition of the conformational selection
mechanism should focus on the sampling of bound conformations of key interface
residues.
References
[1] J. Janin, "Protein-protein docking tested in blind predictions: the CAPRI experiment", Mol Biosyst, 6,
12, 2351-62, 2010.
[2] T. M. Cheng, T. L. Blundell, and J. Fernandez-Recio, "pyDock: electrostatics and desolvation for
effective scoring of rigid-body protein-protein docking", Proteins, 68, 2, 503-15, 2007.
[3] C. Pallara, B. Jimenez-Garcia, L. Perez-Cano, M. Romero-Durana, A. Solernou, S. Grosdidier, C. Pons,
I. H. Moal, and J. Fernandez-Recio, "Expanding the frontiers of protein-protein modeling: from
docking and scoring to binding affinity predictions and other challenges", Proteins, 81, 12, 2192-200,
2013.
[4] C. Pons, S. Grosdidier, A. Solernou, L. Perez-Cano, and J. Fernandez-Recio, "Present and future
challenges and limitations in protein-protein docking", Proteins, 78, 1, 95-108, 2010.
[5] N. Andrusier, E. Mashiach, R. Nussinov, and H. J. Wolfson, "Principles of flexible protein-protein
docking", Proteins, 73, 2, 271-89, 2008.
STABILIZATION OF THE INTEGRIN β4-PLECTIN INTERACTION
WITH THIOL REACTIVE FLUORESCENT COMPOUNDS
José A. Manso1, Stephane Boivin2 and José M. de Pereda1
1
Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas University of Salamanca, Salamanca, Spain
pereda@usal.es
2
European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Hamburg, Germany
Hemidesmosomes are junctional complexes that mediate the stable adhesion of epithelial cells to
the basement membrane (1-2). Integrin α6β4 and plectin are two essential components of the
hemidesmosomes. Plectin binds to the cytoplasmic domain of the integrin β4 subunit; the primary
interaction occurs between the actin-binding domain (ABD) of plectin and a region of β4
consisting of two fibronectin type III domains (FnIII-1,2) and a small sequence of the adjacent
region termed the connecting segment. We have previously elucidated the crystal structure of the
primary α6β4-plectin complex (3). Disruption of the integrin β4-plectin binding interface is linked
to the development of epidermolysis bullosa (EB), an inherited disease characterized by fragility
and blistering of the skin. Two missense mutations in β4, R1225H and R1281W, inhibit the
formation of the complex and have been detected in patients with non-lethal form of EB with
pyloric atresia (3-5). To date no compounds that modulate the dynamics of hemidesmosomes are
known. In this work, we have combined Cys-scanning mutagenesis and labeling with thiolreactive fluorescent compounds to explore the stabilization of the β4-plectin interaction. Six
plectin ABD mutants in which a Cys was engineered near the β4-binding interface were created.
The affinity for β4 of the plectin mutants, each labeled with 9 different probes, was determined
by fluorescence assays. The binding of seven labeled-mutants of plectin to β4 was further
characterized by isothermal titration calorimetry. We have identified specific positions in plectin
on which some probes stabilize the integrin β4-plectin interaction. The largest stabilization effect
was observed when the plectin ABD was derivatized with probes that contain naphthalene or
pyrene groups. The labeling positions that stabilize the interaction are located near a pocket at the
rim of the β4-plectin interface. Stabilization of the β4-plectin interaction rescued binding in the
presence of the β4 mutations R1225H and R1281W that are linked to the development of EB. Our
results pave the way to search for compounds that stabilize the hemidesmosomes. In addition, this
approach could be generally applied for probing other protein-protein interactions.
Acknowledgements: This work has been supported by the Junta de Castilla y León (grant CSI181A12 to
JMdP). The research leading to these results has received funding from the European Community's Seventh
Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). JAM was
recipient of a Postdoctoral Fellowship of the “Programa de Captación de Talento” (Botin Foundation). We
would like to extend special thanks to Rob Meijers and the Sample Preparation and Characterization (SPC)
facility at EMBL Hamburg for support and access to instruments.
References
[1] J.M. de Pereda, E. Ortega, N. Alonso-García, M. Gómez-Hernández, A. Sonnenberg, "Advances and perspectives
of the architecture of hemidesmosomes", Cell Adh. Migr., 3, 4, 361−363, 2009.
[2] Sandy H.M. Litjens, José M de Pereda, Arnoud Sonnenberg, "Current insights into the formation and breakdown
of hemidesmosomes", Trends Cell Biol., 16, 7, 376−383, 2006.
[3] José M de Pereda, M Pilar Lillo, Arnoud Sonnenberg, "Structural basis of the interaction between integrin α6β4
and plectin at the hemidesmosomes”, EMBO J., 28, 8, 1180−1190, 2009.
[4] J. Koster, I. Kuikman, M. Kreft, A. Sonnenberg, "Two different mutations in the cytoplasmic domain of the
integrin β4 subunit in nonlethal forms of epidermolysis bullosa prevent interaction of β4 with plectin", J. Invest.
Dermatol., 117, 6, 1405-1411, 2001.
[5] A. Nakano, L. Pulkkinen, D. Murrell, J. Rico, A. W Lucky, M. Garzon, C. A Stevens, S. Robertson, E. Pfendner,
J. Uitto “Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the β4 integrin gene (ITGB4)
and genotype/phenotype correlations”, Pediatr. Res, 49, 618–626, 2001.
HOW ALLOSTERIC CONTROL OF STAPHYLOCOCCUS AUREUS
PENICILLIN-BINDING PROTEIN 2A ENABLES METHICILLINRESISTANCE AND PHYSIOLOGICAL FUNCTION
Lisandro H. Oteroa, Alzoray Rojas-Altuvea, Leticia I. Llarrullb, Cesar Carrasco-Lópeza,
Malika Kumarasirib, Elena Lastochkinb, Jennifer Fishovitzb, Matthew Dawleyb, Dusan Hesekb,
Mijoon Leeb, Jarrod W. Johnsonb, Jed F. Fisherb, Mayland Changb, Shahriar Mobasheryb
and Juan A. Hermosoa
a
Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física "Rocasolano",
CSIC, Serrano 119, 28006-Madrid, Spain
b
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA
The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical
resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus
(MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in
separate domains the transglycosylase and transpeptidase activities required for the
biosynthesis of the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the
transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of
irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the
PBP2a of MRSA is resistant to β-lactamacylation. The inability to contain MRSA
infection with β-lactam antibiotics is a continuing public health concern. We reported (1)
the identification of an allosteric binding domain (a remarkable 60 Å distant from the
DD-transpeptidase active site) discovered by crystallographic analysis of a soluble
construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational
change culminates in the opening of the active site to permit substrate entry. This same
crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid
(a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and
ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an antiMRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus
predisposing PBP2a to inactivation by a second β-lactam molecule, opens an
unprecedented realm for β-lactam antibiotic structure-based design. We will describe in
the talk the recent advances in this field.
(1) Otero et al. Proceedings of the National Academy of Sciences (2013) 110, 1680816813.
STRUCTURAL BASIS FOR THE MODULATION OF TUBULIN
BY ANTITUMORAL DRUGS
J. Fernando Díaz1, Andrea Prota2 and Michel O. Steinmetz2
1
Centro de Investigaciones Biológicas (CIB-CSIC), Physical and Chemical Biology, Madrid, Spain
fer@cib.csic.es
2
Paul Scherrer Institut, Laboratory of Biomolecular Research, Villigen, Switzerland
Tubulin modulators work by altering the activation state of tubulin, either by deactivating the active GTPbound molecule or activating the otherwise inactive GDP-bound molecule. We have successfully
crystallized the ternary complexes formed between the Tubulin2-RB3 and several microtubule modulating
drugs in three different regulatory sites, this of the paclitaxel site (1), the lauli/peloruside site (2) and a
newly described site to which maytansine, rhizoxin or the Phase I drug PM060184 (3) bind finding the
structural determinants modified in tubulin by these drugs and responsible for the activation (paclitaxel,
laulimalide/peloruside sites) or deactivation (maytansine site).
Ligands of the paclitaxel site bind to a hydrophobic cavity in inner lumen of the microtubules (4). Despite
the importance of paclitaxel for medical applications and basic research, their molecular mechanisms of
action on tubulin and microtubules remain elusive. We determined high-resolution crystal structures of tubulin in complex with two unrelated MSAs, zampanolide and epothilone A (5). Both compounds were
bound to the taxane pocket of -tubulin and used their respective side chains to induce structuring of the
M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts in microtubules, these
findings explain how taxane-site MSAs promote microtubule assembly and stability.
Secondly we have found using X-ray crystallography that laulimalide and peloruside A bind to a unique
non-taxane site on -tubulin and use their respective macrolide core structures to interact with a second
tubulin dimer across protofilaments (6). At the same time, they allosterically stabilize the taxane-site Mloop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin
with laulimalide/peloruside A and epothilone A are also solved.
Finally we have found that Maytansine rhizoxin or the Phase I drug PM060184 bind to an unprecedented
site on ‐tubulin and blocks the formation of longitudinal tubulin interactions in microtubules.
Our results explain the molecular mechanism of action of clinically relevant microtubule modulating
agents. They further provide a structural basis for the rational design of highly potent microtubule
modulating agents, opening a possible route for the development of next‐generation drugs for the treatment
of cancer.
Acknowledgements: This work was supported by grants from the Ministerio de Economía y Competitividad
(BIO2010‐16351 to JFD) and the Comunidad Autónoma de Madrid (S2010/BMD‐2457 to JFD) and by a grant
from the Swiss National Science Foundation (310030B_138659; to MOS)
References:
1. Parness, J., and Horwitz, S. B. (1981) Taxol binds to polymerized tubulin in vitro, J Cell Biol 91, 479-487.
2. Pryor, D. E., O'Brate, A., Bilcer, G., Díaz, J. F., Wang, Y., Kabaki, M., Jung, M. K., Andreu, J. M., Ghosh, A. K., Giannakakou,
P., and Hamel, E. (2002) The microtubule stabilizing agent laulimalide does not bind in the taxoid site, kills cells resistant to
paclitaxel and epothilones, and may not require its epoxide moiety for activity, Biochemistry 41, 9109-9115.
3. Pera, B., Barasoain, I., Pantazopoulou, A., Canales, A., Matesanz, R., Rodriguez-Salarichs, J., Garcia-Fernandez, L. F., Moneo,
V., Jimenez-Barbero, J., Galmarini, C. M., Cuevas, C., Penalva, M. A., Diaz, J. F., and Andreu, J. M. (2013) New Interfacial
Microtubule Inhibitors of Marine Origin, PM050489/PM060184, with Potent Antitumor Activity and a Distinct Mechanism, ACS
Chemical Biology 8, 2084-2094.
4. Nogales, E., Whittaker, M., Milligan, R. A., and Downing, K. H. (1999) High-resolution model of the microtubule, Cell 96, 7988.
5. Prota, A. E., Bargsten, K., Zurwerra, D., Field, J. J., Diaz, J. F., Altmann, K. H., and Steinmetz, M. O. (2013) Molecular
mechanism of action of microtubule-stabilizing anticancer agents, Science 339, 587-590.
6. Prota, A. E., Bargsten, K., Northcote, P., M., M., Altmann, K. H., Miller, J. H., Díaz, J. F., and Steinmetz, M. (2014) Structural
basis of microtubule stabilization by laulimalide/peloruside and their synergy with taxane site anticancer drugs, Angew Chem Int
Ed Engl 53(6):1621-5.
7.- Prota, A.E, Bargsten, K., Diaz, J.F., May Marsh, M. Cuevas, C., Liniger, M., Neuhaus, C., Andreu, J.M. Altmann K.-H. and
Steinmetz, M. O. A new tubulin‐binding site and pharmacophore for microtubule‐destabilizing anticancer drugs. Submitted
ORAL COMMUNICATIONS
S6: Cell Biophysics
S6.OP1. Complete characterization of proteins using ÄKTA and their link with Biacore
and MicroCal ITC/DSC.
Enrique García (GE Healthcare Life Sciences).
S6.OP2. Nosological imaging of glioblastoma response to therapy. (P28)
Magdalena Ciezka, Teresa Delgado-Goñi, Sandra Ortega-Martorell, Ivan Olier,
Margarida Julià-Sape, Ana Paula Candiota, Paulo J.G. Lisboa and Carles Arús.
S6.OP3. Binding of the bacterial RepA-WH1 prionoid to model lipid vesicles. (P39)
Cristina Fernández, Mercedes Jiménez, Germán Rivas and Rafael Giraldo.
S6.OP4. Magnetic nanoparticles and their interaction with cancer cells analyzed by
integrative microscopy and spectroscopy. (P61)
Francisco Javier Chichón, José Javier Conesa, Michele Chiappi, Eva Pereiro, María
Josefa Rodríguez and José L. Carrascosa.
COMPLETE CHARACTERIZATION OF PROTEINS USING ÄKTA
AND THEIR LINK WITH BIACORE AND MICROCAL ITC/DSC
Enrique García
GE Healthcare Life Sciences
With the ability to purify virtually any biomolecule, ÄKTA protein purification systems
can handle the simplest and the toughest of challenges. The platform covers all major
chromatographic techniques, from the research laboratory to process development and
manufacturing. ÄKTA systems are operated by the intelligent and scalable UNICORN
software, which makes it easy to control every stage of your purification process.
Together with our broad range of pre packed chromatography columns and
chromatography media, ÄKTA systems and UNICORN software provide a complete
solution for great results. During the presentation, we introduce the new systems: ÄKTA
start and ÄKTA pure and their link with Biacore and MicroCal ITC/DSC for the complete
characterization of proteins.
NOSOLOGICAL IMAGING OF GLIOBLASTOMA RESPONSE TO THERAPY
Magdalena Ciezka1,2,3, Teresa Delgado-Goñi4, Sandra Ortega-Martorell5,2, Ivan Olier6,
Margarida Julià-Sape2,1,3, Ana Paula Candiota2,1,3, Paulo J.G. Lisboa5, Carles Arús1,2,3
mciezka@gabrmn.uab.es
1
Department de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Edifici Cs,
Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
2
Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina
(CIBER-BBN), Spain
3
Institut de Biotecnologia i de Biomedicina, Cerdanyola del Vallès, Spain
4
The Institute of Cancer Research, London, United Kingdom
5
Department of Mathematics and Statistics, Liverpool John Moores University,
Liverpool, United Kingdom
6
Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom
Introduction: Glioblastomas (GBM) are high-grade brain tumours with poor survival
and bad prognosis. There are no robust methods for early assessment of therapy response
available. The rich information contained in magnetic resonance (MR) signals (i.e.
spectroscopy, MRS/ spectroscopic imaging, MRSI) makes them ideally suited for the
application of pattern recognition (PR) techniques. Source extraction analysis can provide
the identification of tissue type-specific sources and generate color-coded maps,
displaying the spatial accumulation of metabolites or tumour delimitation [1]. Dimethyl
sulfoxide (DMSO) has been reported as a potential contrast agent to evaluate GBM
response to therapy [2].
Purpose: To investigate the possibility of non-invasive therapy response assessment in
GBM through imaging of the basal and DMSO-perturbed MRSI (PE-MRSI) in preclinical
models.
Materials and Methods: C57BL/6 female mice (n=91) were inoculated with GL261
glioma cells as in [3]. MR studies were carried out at 7T, with isoflurane anaesthesia (1.52%) at 37°C. The therapy consisted in three TMZ cycles [4]. Magnetic resonance images
(MRI, T2w) (TR/TEeff 4200/36ms) were acquired for tumour volume measurement.
Treated mice were studied by PE-MRSI with DMSO. A reference T2w MRI and a 14ms
TE control were acquired before DMSO injection. Parameters for MRSI were as in [3].
Data were post-processed with 3DiCSI and MatLab to generate time-course maps of
DMSO heights and semi-supervised source-based maps [5].
Results: The sources extracted from a grid of voxels in a region of interest were able to
discriminate between GL261 tumours actively proliferating and tumours responding to
therapy, based on their metabolome pattern changes recorded by MRSI, before tumour
growth arrest was observed by MRI (Figure 1). Colour-coded nosological images
obtained throughout the course of therapy allowed convenient tracking of response
changes.
Conclusion: Source extraction and nosological imaging can serve as a valuable noninvasive tool in preclinical analysis for therapy response and monitoring.
Figure 1. A) Tumour volume change in the control group (in red) of GL261 tumour bearing mice (n=16)
and one representative TMZ-treated C819 mouse (in blue). Rectangles in light green highlight the 3 TMZ
cycles. B) Representative T2w MR images (top) and nosologic color-coded maps (bottom) corresponding
to mouse C819 and MRSI grid prior to DMSO injection at chosen time points (black triangles in A)). Blue
voxels are assigned, using the semi-supervised method, to normal brain parenchyma, red voxels to
untreated/non-responding tumour and green voxels to treated responding tumour. Tumour boundaries (T 2w
hiperintensity-derived) are marked with a white dotted line.
Acknowledgements: This work was funded by MINECO grant MARESCAN (SAF 2011-23870) and
Centro de Investigación Biomédica en Red - Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN,
[http://www.ciber-bbn.es/en]), an initiative of the Instituto de Salud Carlos III (Spain) co-funded
by EU FEDER. Magdalena Ciezka holds a FI-DGR grant from the Generalitat de Catalunya.
References
1.
Ortega-Martorell, S., et al., Convex non-negative matrix factorization for brain tumor delimitation
from MRSI data. PLoS One, 2012. 7(10): p. e47824.
2.
Delgado-Goni, T., et al., Dimethyl sulfoxide (DMSO) as a potential contrast agent for brain
tumors. NMR Biomed, 2013. 26(2): p. 173-84.
3.
Simoes, R.V., et al., 1H-MRSI pattern perturbation in a mouse glioma: the effects of acute
hyperglycemia and moderate hypothermia. NMR Biomed, 2010. 23(1): p. 23-33.
4.
Delgado-Goni, T., et al., DMSO-based contrast for monitoring GBM response to therapy. Magn
Reson Mater Phy MAGMA, 2011. 24(S1): p. 119-120.
5.
Ortega-Martorell S et al. A novel semi-supervised methodology for extracting tumor type-specific
MRS sources in human brain data. PLoS One. 2013 Dec 23;8(12):e83773.
BINDING OF THE BACTERIAL RepA-WH1 PRIONOID
TO MODEL LIPID VESICLES
Cristina Fernández, Mercedes Jiménez, Germán Rivas, and Rafael Giraldo
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Madrid, Spain.
Email: cfernandez@cib.csic.es
We have recently reported that engineering RepA-WH1, a bacterial DNA-toggled protein
conformational switch (dWH1  mWH1) sharing some analogies with nucleic acidpromoted PrPc  PrPSc replication [1], constitutes a suitable synthetic model system to
study protein amyloidosis in bacteria [2, 3]. Although amyloidogenesis has been the focus
of intense research, the origin of the amyloid toxicity remains unclear. One proposed
mechanism of cytotoxicity is lipid membrane permeabilization.
In this work we have studied the aggregation of the bacterial RepA-WH1 prionoid in the
presence of cytomimetic model systems (large and giant unilamellar lipid vesicles, LUVs,
and GUVs respectively) [4]. We have observed that the interaction of RepA-WH1 with
membranes can catalyze aggregation. Confocal microscopy images of protein
encapsulated into GUVs show association and aggregation of the protein preferentially to
lipid vesicles containing acidic phospholipids. We have also observed that RepA-WH1
elicits membrane disruption using a dye release assay on LUVs [5]. The extent of leakage
was dependent on protein concentration.
We have been able to directly measure the process of membrane permeation and leakage
by time-elapsed imaging of dye filled GUVs upon the addition of protein. This process is
fast and over the course of the experiment most of the vesicles remain intact, suggesting
the assembly of defined pores by RepA-WH1. Knowledge of the effect of the RepA-WH1
prionoid on membrane integrity, will provide insight into the basis for cell death caused
by amyloid proteins.
References
[1] Jerson L. Silva, Luis Mauricio T. R. Lima, Debora Foguel, Yraima Cordeiro, “Intriguing nucleic-acidbinding features of mammalian prion protein”, Trends Biochem. Sci., 33, 3, 132-140, 2008.
[2] Rafael Giraldo, Susana Moreno-Díaz de la Espina, M. Elena Fernández-Tresguerres, Fátima GassetRosa, “RepA-WH1 prionoid: a synthetic amyloid proteinopathy in a minimalist host”, Prion, 5, 6064, 2011.
[3] M. Elena Fernández-Tresguerres, Susana Moreno-Díaz de la Espina, Fátima Gasset-Rosa, Rafael
Giraldo, “A DNA-promoted amyloid proteinopathy in Escherichia coli”, Mol. Microbiol. 77, 14561469, 2010.
[4] Sara M. Butterfield, Hilal A. Lashuel, “Amyloidogenic protein-membrane interactions: mechanistic
insight from model systems”, Angew Chem Int Ed. 49, 5628-5654, 2010.
[5] Bart D. van Rooijen, Mireille M.A.E. Claessens, Vinod Subramaniam, “Lipid bilayer disruption by
oligomeric α–synuclein depends on bilayer charge and accessibility of the hydrophobic core”,
Biochim. Biophys. Acta, 1788, 1271-1278, 2009.
MAGNETIC NANOPARTICLES AND THEIR INTERACTION WITH
CANCER CELLS ANALYZED BY INTEGRATIVE
MICROSCOPY AND SPECTROSCOPY
Francisco Javier Chichón1, José Javier Conesa1, Michele Chiappi2, Eva Pereiro2,
María Josefa Rodríguez1 and José L. Carrascosa1,3
1
Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049 Madrid, Spain.
2
ALBA Synchrotron Light Source. MISTRAL Beamline - Experiments division.
08290 Cerdanyola del Vallès, Barcelona, Spain.
3
Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia),
Cantoblanco, 28049 Madrid, Spain.
We have studied the interaction, internalization and accumulation of dimercaptosuccinic
acid-coated superparamagnetic iron oxide nanoparticles (DMSA-SPION), with average
diameter of 15 nm and negative surface charge, in MCF-7 breast cancer cells. Cell
cultures were incubated with 0.25 mg Fe ml-1 DMSA-SPIONs from 0 to 24 h.
Light microscopy, transmission electron microscopy (TEM) and Soft X-ray cryotomography were combined to characterize the interaction and accumulation of DMSASPION in MCF-7 cells, as well as the reorganization of the intracellular environment due
to the nanoparticle uptake. The correlation of these different techniques allowed to
visualize, at nanometric three-dimensional resolution, the whole cell without chemical
fixation or staining agents. Correlative microscopy was used to locate the cells containing
nanoparticles accumulated in endosomes. Reconstructed volumes show the SPIONcontaining endosomal accumulation near the Golgi area, close to the nucleus.
Moreover, we acquired tilted series of the samples at different energies for tomographic
reconstructions. We used 700 eV (before the iron L3 edge) and 707 eV (on the L3 iron
edge) to localize in 3D the presence of iron. These series were aligned and reconstructed
to obtain the 3D distribution of iron particles within the sample.
Acknowledgements: These experiments were performed at Mistral beamline at ALBA Synchrotron
Light Facility with the collaboration of ALBA staff. We must acknowledge too Cedric Messaodi for
the software support from the Sergio Marco laboratory at Curie Institute.
ORAL COMMUNICATIONS
S7: Biophysics of Nucleic Acids
S7.OP1. Force-dependent melting of supercoiled DNA at thermophilic temperatures.
(P03)
Eric Galburt, Eric Tomko, Tom Stump and Ana Ruiz Manzano.
S7.OP2. Discovery of selective ligands for telomeric RNA G-quadruplexes (TERRA)
through 19F-NMR based fragment screening. (P41)
Miguel Garavís Cabello, B. López-Méndez, A. Somoza, J. Oyarzabal, C. Dalvit,
A. Villasante, R. Campos-Oliva and C. González.
S7.OP3. Single-molecule mechanical characterization of the HmtSSB binding
properties to ssDNA. (P63)
José A. Morin, Laurie S. Kaguni and Borja Ibarra.
S7.OP4. Magnetic tweezers studies of the type IA topoisomerase RepC.
Carolina Carrasco, César L. Pastrana, Parvez Akhtar, Sanford H. Leuba, Saleem A. Khan
and Fernando Moreno-Herrero.
FORCE-DEPENDENT MELTING OF SUPERCOILED DNA
AT THERMOPHILIC TEMPERATURES
Eric A. Galburt, Eric J. Tomko, Tom Stump and Ana Ruiz Manzano
Washington University School of Medicine, St. Louis, MO, USA
egalburt@biochem.wustl.edu
Local DNA opening plays an important role in DNA metabolism as the double-helix must
be melted before the information contained within may be accessed. Cells finely tune the
torsional state of their genomes to strike a balance between stability and accessibility. For
example, while mesophilic life forms maintain negatively superhelical genomes,
thermophilic life forms use unique mechanisms to maintain relaxed or even positively
supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to
quantify the force-dependent equilibrium between DNA melting and supercoiling at high
temperatures populated by Thermophiles. We show that negatively supercoiled DNA
denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work
demonstrates the ability to monitor DNA supercoiling at high temperature and opens the
possibility to perform magnetic tweezers assays on thermophilic systems. The data allow
for an estimation of the relative energies of base-pairing and DNA bending as a function
of temperature and support speculation as to different general mechanisms of DNA
opening in different environments. Lastly, our results imply that average in vivo DNA
tensions range between 0.3 - 1.1 pN.
Acknowledgements: This work has been sponsored by Washington University in Saint Louis
References
[1] Eric Galburt, Eric Tomko, Tom Stump, and Ana Ruiz Manzano, “Force-dependent Melting of
Supercoiled DNA at Thermophilic Temperatures”, Biophysical Chemistry, 187-188, 23-28, 2014.
DISCOVERY OF SELECTIVE LIGANDS FOR TELOMERIC RNA
G-QUADRUPLEXES (TERRA) THROUGH 19F-NMR BASED
FRAGMENT SCREENING
M. Garavís1, B. López-Méndez2, A. Somoza3, J. Oyarzabal2, C. Dalvit2,
A. Villasante4, R. Campos-Oliva2 and C. González1
1
Instituto de Química Física 'Rocasolano', CSIC. Serrano 119, 28006 Madrid (Spain)
2
Spectroscopy and NMR Unit & Experimental Therapeutics Programme,
Spanish National Cancer Research Center (CNIO), Melchor Fernández Almagro 3, 28029 Madrid (Spain)
3
IMDEA Nanociencia and CNB-CSIC-IMDEA Nanociencia Associated Unit
‘‘Unidad de Nanobiotecnologia’’ Cantoblanco, 28049 Madrid (Spain)
4
Centro de Biología Molecular ‘‘Severo Ochoa’’ (CSIC-UAM),
Universidad Autónoma de Madrid, Madrid (Spain)
Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral
target. Here we report the first successful application of 19F-NMR fragment-based
screening to identify chemically diverse compounds that bind to an RNA molecule such
as TERRA. We have built a library of 355 fluorinated fragments, and checked their
interaction with a long telomeric RNA as a target molecule. The screening resulted in the
identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with
TERRA was confirmed by 19F- and 1H-NMR as well as by CD melting experiments. We
have also explored the selectivity of the ligands for RNA G-quadruplexes, and found that
some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA
and, most importantly, favor the propeller-like parallel conformation in telomeric DNA
G-quadruplexes. This suggests a selective recognition of this particular quadruplex
topology and that different ligands may recognize specific sites in propeller-like parallel
G-quadruplexes. Such features make some of the resulting binders promising lead
compounds for fragment based drug discovery.
Acknowledgements: MICINN (CTQ2010-21567-C02-02, BFU2011-30295-C02-01, SAF201015440), Comunidad Autónoma de Madrid (S2010-BMD-2457, BIPEDD2), Institutional grant
from the Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa”). MG
was supported by the FPI-fellowship BES-2009-027909.
Reference
[1] M. Garavís et al., “Discovery of selective ligands for telomeric RNA G-quadruplexes (TERRA) through
19
F-NMR based fragment screening”, ACS Chem Biol, in press, 2014 (PMID: 24837572).
SINGLE-MOLECULE MECHANICAL CHARACTERIZATION OF
THE HmtSSB BINDING PROPERTIES TO ssDNA.
José A. Morin1, Laurie S. Kaguni2 and Borja Ibarra1
1
Imdea Nanociencia, Faraday, Nº 9 Ciudad Universitaria de Cantoblanco, 28049. Madrid (España)
2
Department of Biochemistry and Molecular biology, Michigan State University,
319 Biochemistry Building Michigan State University, East Lansing, MI 48824-1319, USA.
Mitochondrial DNA (mtDNA) encodes for most of the components of the cellular
electron transport chain. Mutations on mtDNA have been associated with respiratory
chain dysfunction, which in turn causes rare diseases in plants and humans. Therefore,
faithful replication of mtDNA is essential for the correct functioning of the cell. The
human mitochondrial single-stranded DNA binding protein (HmtSSB) plays a critical
role at the mtDNA replication fork, coordinating the interactions between single-stranded
DNA (ssDNA), the DNA polymerase γ and the mtDNA helicase [1]. However, little is
known about the real time kinetics of the HmtSSB interaction with the ssDNA and the
structural and mechanical properties of the HmtSSB-coated ssDNA polymer; which is
essential information to understand the role of the HmtSSB on DNA metabolism. Using
optical tweezers ([2]) we have developed a single molecule manipulation assay that
addresses these questions. Our data show that the mechanical properties of ssDNA change
dramatically in the presence of HmtSSB and they strongly depend on both the monovalent
salt (0.05 to 0.3 M NaCl) and protein concentrations (5 to 200 nM) suggesting that
HmtSSB interacts with ssDNA in two different modes [3]. Moreover, force pulling
experiments designed specifically to test the stability of the HmtSSB-coated polymer
show that HmtSSB units associate with each other in a cooperative fashion, whose
strength also depends on ionic conditions and protein concentration. Consistent with this
observation, real time polymerization kinetics exhibit two distinct behaviors,
characterized by the apparent cooperativity, that interchange continuously as the ratio of
salt to protein concentration is tuned.
References
[1] M. Falkenberg, N. Larsson and C. Gustafsson, “DNA replication and transcription in mammalian
mitochondria.”, Annu Rev Biochem, 76, 679-699, 2007.
[2] S. B. Smith, Y. Cui and C. Bustamante, “Optical-trap force transducer that operates by direct
measurement of light momentum.”, Methods Enzymol, 361, 134-162, 2003.
[3] T. M. Lohman and M. E. Ferrari, “Eschericçhia coli single-stranded DNA-binding protein: multiple
DNA-binding modes and cooperativities.”, Annu Rev Biochem, 63, 527-570, 1994.
MAGNETIC TWEEZERS STUDIES OF THE
TYPE IA TOPOISOMERASE RepC
Carolina Carrasco 1, César L. Pastrana 1, Parvez Akhtar 2, Sanford H. Leuba 2,
Saleem A. Khan 2, and Fernando Moreno-Herrero1
1
Centro Nacional de Biotecnología, CSIC, Campus UAM, Darwin 3, 28049 Cantoblanco, Madrid, Spain
2
Dept. of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, EEUU
A precise and time-dependent control of the level of DNA supercoiling is essential in
many cellular processes such as replication, recombination and transcription. This task is
carried out by the action of topoisomerases by introducing transient single or doublestrand breaks in the DNA [1]. We have used Magnetic Tweezers (MT) to study the
enzymatic activity of RepC, a plasmid-encoded type IA topoisomerase from
Staphylococcus aureus [2]. SaRepC is the replication initiator protein of plasmid pT181 and
constitutes a prototype to study rolling-circle replication (RCR) of plasmids in Grampositive bacteria. RepC nicks plasmid DNA at the origin of replication and remains
covalently bound to the DNA through a phosphotyrosine bond while supercoils are
released. Then, RepC ligates the previously formed nick and releases the relaxed DNA
[3]. In MT, a single molecule of DNA is tethered between a micrometer-size magnetic
bead and the surface of a liquid cell. By rotating a pair of magnets above the liquid cell,
different number of supercoils can be induced on torsionally-constrained DNA molecules
[4]. We measured both nicking and re-ligation activities of RepC on supercoiled DNA
containing its nick site. RepC was only active on negatively supercoiled DNA suggesting
that binding to DNA requires the formation of specific DNA structures at the origin
region. Moreover, supercoils were released in a vast majority of cases in a single step.
Characterization of RepC activity constitutes a starting point to study the combined
activities of topoisomerases and helicases in RCR of plasmids.
References
[1] Champoux, J. J. (2001). DNA Topoisomerases: Structure, Function, and Mechanism. Annu Rev
Biochem 70, 635-92.
[2] Koepsel, R. R., et al. (1985). The replication initiator protein of plasmid pTl81 has sequence-specific
endonuclease and topoisomerase-like activities. Proc. Natl. Acad. Sci. USA 82, 6845-6849.
[3] Koepsel, R. R., et al. (1986). Sequence-specific interaction between the replication initiator protein of
plasmid pT181 and its origin of replication. Proc. Natl. Acad. Sci. USA 83, 5484-5488.
[4] Strick T. R., et al. (1998). Behavior of supercoiled DNA. Biophy Journal74, 2016–2028.
ORAL COMMUNICATIONS
S8: Membrane Biophysics: Organization and Dynamics
S8.OP1. Membrane properties of novel 1-deoxyceramides. (P10)
Noemí Jiménez-Rojo, Félix M. Goñi and Alicia Alonso.
S8.OP2. Modelling the interplay between protein and lipid aggregation in supported
membranes. (P65)
Pablo González de Prado Salas, Pedro Tarazona and Marisela Vélez.
S8.OP3. Lipid membrane domains shaped by artificially cholesterol recycling in model
vesicles. (P79)
Iván López Montero, Pilar Lillo and Francisco Monroy.
S8.OP4. Pores of melittin in supported lipid monolayers as observed by AFM. (P88)
Jesús Salgado, Diana Giménez and Orlando L. Sánchez Muñoz.
MEMBRANE PROPERTIES OF NOVEL 1-DEOXYCERAMIDES
Noemi Jiménez-Rojoa*, Aritz B. García-Arribasa, Walt A. Shawb, Jingjing Duanc,
Alfred H. Merrill, Jr. c, Félix M. Goñia, Alicia Alonsoa
a
Unidad de Biofísica (Centro Mixto CSIC, UPV/EHU), and Departamento de Bioquímica y
Biología Molecular, Universidad del País Vasco (UPV/EHU).
b
Avanti Polar Lipids, Inc. Alabaster, AL 35214 U.S.A.
c
School of Biology and Petite Institute for Bioengineering and Bioscience,
Georgia Institute of Technology, Atlanta, Georgia, U.S.A.
Serine palmitoyl transferase (SPT) is the enzyme that catalyses the first and rate limiting
step of the de novo synthesis of sphingolipids which consists of the condensation of
serine with palmitoy-CoA to produce 3-ketosphinganine. However it has been recently
demonstrated that this enzyme can also condense the non-canonical aminoacids alanine
and glycine to give rise to the novel 1-deoxysphingolipids, until present two
underappreciated lipid species. Previous data have suggested that the non-canonical 1deoxysphingolipids are biologically active as a dysregulation of their production results
in the human disease hereditary sensory autonomic neuropathy type 1 (HSAN1) (1). At
present there is no data available about the biophysical properties of these novel noncanonical 1-deoxysphingolipids. The complex biophysical properties observed in this
work suggest that these lipids may have dramatic effects in the structure and integrity of
biological membranes, particularly the endoplasmic reticulum membrane where they are
synthesized, leading, as a consequence, to ER stress. This may implicate these lipids in
important processes for cell physiology such as autophagy-mediated cell death, closely
related to ER dysfunction. Thus, their complex physico-chemical properties could be at
the origin of their pathological implications and this study opens a number of
possibilities for a function at the membrane level for these novel 1-deoxysphingolipids.
In the present contribution, different approaches have been applied to study the
behaviour of some deoxy (1-dcer), dihydrodeoxy (1-ddcer) and dihydrodeoxymethylceramides (1-ddmetcer). The thermotropic behavior of pure 1-deoxyceramides and of
SM-deoxyceramide mixtures is described, together with the SM-deoxyceramide
interactions in monolayers, as well as the behaviour of these lipids in more complex
lipid mixtures.
Acknowledgments. This work has been supported in part by grants from the Spanish
Ministerio de Economía (BFU 2012-36241; BFU 2011-28566) and the Basque
Government (IT838-13; IT849-13).
Reference
[1] Penno A, Reilly MM, Houlden H, Laura M, Rentsch K, Niederkofler V, Stoeckli ET, Nicholson G,
Eichler F, Brown RH, Jr., von Eckardstein A, Hornemann T. Hereditary sensory neuropathy type 1 is caused
by the accumulation of two neurotoxic sphingolipids. J Biol Chem. (2010) 285: 11178-11187.
MODELLING THE INTERPLAY BETWEEN PROTEIN AND
LIPID AGGREGATION IN SUPPORTED MEMBRANES
Pablo González de Prado Salas1, Pedro Tarazona2 and Marisela Vélez3
1
Universidad Autónoma de Madrid, Madrid, Spain.
pablo.gonzalezdeprado@uam.es
2
Centro de Investigación de Física de la Materia Condensada and Instituto de Ciencia de Materiales
Nicolás Cabrera, Universidad Autónoma de Madrid, Madrid, Spain
3
Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain
We present a theoretical model that deals with the complex interplay between lipid
segregation and the self-aggregation of lipid-attached proteins. The model, in contrast to
previous models that consider proteins only as passive elements affecting the lipid
distribution, describes the system including dynamic interactions between protein
monomers, the interactions between lipid components, and also a mixed term considering
both protein-lipid interactions.
This model is an extension of a previous one1,2 used to study the dynamics of the selfaggregating cytoskeletal protein FtsZ, and has been expanded to explain experimental
results performed on a well-defined system that includes two elements: FtsZ proteins and
a lipid bilayer with two lipid components. The model can reproduce the observed
differences determined by the orientation of the filaments on the surface, indicating that
all contributions to filament formation, including the interplay between torsion and an
specific anchoring, are needed to account for the observations. Furthermore, it points out
that lipid segregation can affect the length and curvature of protein filaments and that the
dynamic behaviour of the lipids and proteins might have different time scales, giving rise
to “memory effects”.
This simple model that considers a dynamic protein assembly on a fluid and active lipid
surface can be easily extended to other biologically relevant situations in which the
interplay between protein and lipid aggregation are both needed to fully describe the
system.
Acknowledgements: We acknowledge financial support by the Spanish Ministerio de Ciencia e Innovación
(Grant No. FIS2010-22047-C05), the Comunidad Autónoma de Madrid under program MODELICO
(Grant No. S2009/ESP-1691) and the Ministerio de Educación, Cultura y Deporte (FPU fellowship
program).
References
[1] Pablo González de Prado Salas, Mario Encinar, Marisela Vélez, Pedro Tarazona, “FtsZ protein on
bilayer membranes: Effects of specific lateral bonds”, Soft Matter, 9 (26), 6072–6079, 2013.
[2] Pablo González de Prado Salas, Ines Hörger, Fernando Martín-García, Jesús Mendieta, Álvaro Alonso,
Mario Encinar, Paulino Gómez-Puertas, Marisela Vélez, Pedro Tarazona, “Torsion and curvature of
FtsZ filaments”, Soft Matter, 10 (12), 1977–1986, 2014.
LIPID MEMBRANE DOMAINS SHAPED BY ARTIFICIALLY
CHOLESTEROL RECYCLING IN MODEL VESICLES
Iván López-Montero1, Pilar Lillo2, and Francisco Monroy1
1
Departamento de Química Física I, Universidad Complutense, 28040 Madrid, Spain
2
Instituto de Química –Física Rocasolano, CSIC, 28006 Madrid, Spain
Lipid mixtures of phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol (chol)
can lead to microscopic lipid phase separation on membrane model systems. This
observation suggests the existence of functional lipid rafts in living cells. However, cell
membranes are not static entities but affected by a continuous membrane recycling, which
could reduce the average domain radius in the tens of nm scale. Here, we study the size
of lipid domains in model vesicles under the action of cholesterol recycling. For this
purpose, lipid vesicles made of POPC/EggSM/Chol and DOPC/EggSM/Chol (at 5/2/3
and 3.5/4/2.5 molar ratios respectively) were fabricated in the presence of progressively
increasing concentrations of cholesterol-loaded and -unloaded cyclodextrins (up to 50
M), able to uptake and release cholesterol molecules from/to lipid bilayers. By means
of time-resolved FRET performed on 100 nm size liposomes, we have been able to
measure a reduction of the lo-domain size up to 4% and 13 % for POPC- and DOPCcontaining bilayers respectively.
PORES OF MELITTIN IN SUPPORTED LIPID MONOLAYERS
AS OBSERVED BY AFM
Diana Giménez, Orlando L. Sánchez Muñoz and Jesús Salgado
University of Valencia, Institute of Molecular Science (ICMol), Paterna (Valencia) Spain.
E-mail: jesus.salgado@uv.es
Melittin, a membrane lytic peptide from bee venom is often considered archetypal among
the large and diverse number of pore-foming peptides. It is used as a model for the
understanding of the structure and mechanism of these systems and as a scaffold for the
development of new pore-forming molecules with potential uses in medicine and
biotechnology [1]. However, the structure of melittin in the pores is still unknown and
there is even debate about the existence of these pores and their importance for melittin
function [1,2]. In this work we report the direct observation of melittin-induced pores in
supported lipid monolayers by atomic force microscopy (AFM). Because the interaction
of melittin with lipid bilayers occurs essentially at the interface level, the monolayer
provides an analogous docking site for the peptide. It is therefore a good model for the
initial stages of binding to membranes. We will show that the monolayer systems can also
be considered adequate for trapping relevant structures of the peptide-membrane
complex, including pores. Moreover, the monolayer may represent a way to shift the
thermodynamic balance between melittin bound species and increase the occurrence of
pores compared to their abundance in bilayers. We describe the nanometer scale
properties of these pores, including a rim of protruding material which is likely associated
to the presence of melittin molecules. This study may represent a significant step forward
towards the structural investigation of dynamic pore forming systems using monolayers.
Acknowledgements: This work has been sponsored by MINECO (BFU2010-19118).
References
[1] A.J. Krauson, J. He, W.C. Wimley, “Gain-of-Function Analogues of the Pore-Forming Peptide Melittin
Selected by Orthogonal High-Throughput Screening”, J. Am. Chem. Soc, 134, 12732–12741 (2012).
doi:10.1021/ja3042004.
[2] M.-T. Lee, T.-L. Sun, W.-C. Hung, H.W. Huang, “Process of inducing pores in membranes by
melittin”, Proc. Natl. Acad. Sci. 110, 14243–14248 (2013). doi:10.1073/pnas.1307010110.
Posters XIV Congress of the Spanish Biophysical Society (SBE 2014)
P02
STRUCTURAL CHANGES TRIGGERED BY SOLVENT IN A PEPTIDE
DERIVED FROM PNEUMOCOCCAL CHOLINE-BINDING PROTEIN LytA
Héctor Zamora-Carreras1, Beatriz Maestro2, Jesús M. Sanz2,
Marta Bruix1 and Mª Ángeles Jiménez1
Instituto de Química Física Rocasolano, CSIC, Madrid, Spain. hzamora@iqfr.csic.es
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain
1
2
The use of model peptides derived from natural sequences or designed de novo is a
habitual strategy to better understand the formation and stability of secondary
structures. As important as the sequence itself, a critical factor affecting the dynamics of
peptide structures is the solvent. In this regard, it is well known that some fluorinated
alcohols like trifluoroethanol (TFE) o hexafluoroisopropanol (HFIP) stabilize secondary
structures.[1] In addition, the study of systems whose biological functions imply
interactions with membranes, as the antimicrobial or cell penetrating peptides, has led to
the necessity of reproducing membrane conditions in the solvent. In the case of solution
NMR spectroscopy, the most commonly used membrane models are detergent micelles.
They have been shown to be useful to determine solution structures of peptides, integral
membrane proteins and protein domains.[2]
In this work, we have studied a fourteen-residue peptide (CLyt3) derived from the
choline-binding domain of protein LytA from Streptococcus pneumoniae. This protein
is related to the virulence of the bacteria and it is located in the peptidoglycan envelope
of the cell. The sequence of the peptide forms a β-hairpin structure in the native protein
and it is involved in the binding of choline molecules associated to teichoic and
lipoteichoic acids present in the peptidoglycan.[3][4]
Through an experimental approach based on CD and solution NMR experiments, we
have investigated whether CLyt3 is able to maintain its native β-hairpin structure in
aqueous solution isolated from the rest of the protein. Also, we have examined the
effect of solvent on the peptide structure using the fluorinated alcohol TFE and different
membrane models (DPC and SDS micelles).
Acknowledgements: This work has been sponsored by Spanish MINECO through the project no.
CTQ2011-22514, and the FPI fellowship no. BES-2012-057717.
References
[1] Clara M. Santiveri, David Pantoja-Uceda, Manuel Rico and M. Ángeles Jiménez, “β-hairpin
formation in aqueous solution and in the presence of trifluoroethanol: A 1H and 13C nuclear
magnetic resonance conformational study of designed peptides”, Biopolymers, 79 (3), 150-162,
2005.
[2] Lena Mäler, “Solution NMR studies of cell-penetrating peptides in model membrane systems”,
Advanced Drug Delivery Reviews, 65, 1002-1011, 2013.
[3] Beatriz Maestro, Clara M. Santiveri, M. Ángeles Jiménez and Jesús M. Sanz, “Structural autonomy
of a β-hairpin peptide derived from the pneumococcal choline-binding protein LytA”, Protein
Engineering, Design & Selection, 24 (1-2), 113-122, 2011.
[4] I. Pérez-Dorado, S. Galán-Bartual and J. A. Hermoso, “Pneumococcal surface proteins: when the
whole is greater than the sum of its parts”, Molecular Oral Microbiology, 27, 221-245, 2012.
P03
FORCE-DEPENDENT MELTING OF SUPERCOILED DNA
AT THERMOPHILIC TEMPERATURES
Eric A. Galburt, Eric J. Tomko, Tom Stump and Ana Ruiz Manzano
Washington University School of Medicine, St. Louis, MO, USA
egalburt@biochem.wustl.edu
Local DNA opening plays an important role in DNA metabolism as the double-helix must
be melted before the information contained within may be accessed. Cells finely tune the
torsional state of their genomes to strike a balance between stability and accessibility. For
example, while mesophilic life forms maintain negatively superhelical genomes,
thermophilic life forms use unique mechanisms to maintain relaxed or even positively
supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to
quantify the force-dependent equilibrium between DNA melting and supercoiling at high
temperatures populated by Thermophiles. We show that negatively supercoiled DNA
denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work
demonstrates the ability to monitor DNA supercoiling at high temperature and opens the
possibility to perform magnetic tweezers assays on thermophilic systems. The data allow
for an estimation of the relative energies of base-pairing and DNA bending as a function
of temperature and support speculation as to different general mechanisms of DNA
opening in different environments. Lastly, our results imply that average in vivo DNA
tensions range between 0.3 - 1.1 pN.
Acknowledgements: This work has been sponsored by Washington University in Saint Louis
References
[1] Eric Galburt, Eric Tomko, Tom Stump, and Ana Ruiz Manzano, “Force-dependent Melting of
Supercoiled DNA at Thermophilic Temperatures”, Biophysical Chemistry, 187-188, 23-28, 2014.
P04
CONFORMATIONAL SELECTION MECHANISM IN
PROTEIN-PROTEIN ASSOCIATION: INSIGHTS FROM DOCKING
Chiara Pallara, Manuel Rueda and Juan Fernández-Recio
Joint BSC-IRB Research Programme in Computational Biology, Barcelona Supercomputing Center,
Barcelona, Spain . E-Mail: chiara.pallara@bsc.es
To understand cellular processes at molecular level we need to improve our knowledge
of protein-protein interactions, but determining the atomic structure of many protein
complexes is still challenging. Thus, structural prediction of protein-protein association
is one of the major goals of computational biophysics. Despite methodological advances
in docking protocols, dealing with molecular flexibility is a major bottle-neck, as the
experiment CAPRI (Critical Assessment of PRediction of Interactions) [1] has shown.
Indeed, state-of-the-art rigid-body docking approaches like pyDock [2] show excellent
success rates [3], but have difficulties in cases with large conformational changes upon
binding [4]. For complexes that form via conformational selection mechanism, in which
the unbound state can sample bound conformers, a largely unexplored strategy to include
flexibility in docking predictions would consist on the use of precomputed
conformational ensembles generated from unbound protein structures [5]. Recently we
applied this approach to a series of ubiquitin complexes, in which the use of RDCderived
ensembles significantly improved docking predictions [6]. Here, we have extended this
strategy to the set of 124 cases in Protein-Protein Docking Benchmark 3.0 [7].
Conformational ensembles for the unbound docking partners were automatically
generated by using three different computational approaches, modeling minimization
(MM), molecular dynamics (MD) and normal mode analysis (NMA). To establish the
limits of the approach in optimal conditions, we first used for docking only those
conformers that would be expected to give best results based on their similarity to the
bound structure. Then, for a small sub-set of cases we devised a more realistic protocol
by using all conformers for the docking simulations. The results show that the use of small
conformational ensembles can significantly improve docking predictions in high-affinity,
medium-flexibility complexes. In addition to the relevance for methodology
development, his work shows that the definition of the conformational selection
mechanism should focus on the sampling of bound conformations of key interface
residues.
References
[1] J. Janin, "Protein-protein docking tested in blind predictions: the CAPRI experiment", Mol Biosyst, 6,
12, 2351-62, 2010.
[2] T. M. Cheng, T. L. Blundell, and J. Fernandez-Recio, "pyDock: electrostatics and desolvation for
effective scoring of rigid-body protein-protein docking", Proteins, 68, 2, 503-15, 2007.
[3] C. Pallara, B. Jimenez-Garcia, L. Perez-Cano, M. Romero-Durana, A. Solernou, S. Grosdidier, C. Pons,
I. H. Moal, and J. Fernandez-Recio, "Expanding the frontiers of protein-protein modeling: from
docking and scoring to binding affinity predictions and other challenges", Proteins, 81, 12, 2192-200,
2013.
[4] C. Pons, S. Grosdidier, A. Solernou, L. Perez-Cano, and J. Fernandez-Recio, "Present and future
challenges and limitations in protein-protein docking", Proteins, 78, 1, 95-108, 2010.
[5] N. Andrusier, E. Mashiach, R. Nussinov, and H. J. Wolfson, "Principles of flexible protein-protein
docking", Proteins, 73, 2, 271-89, 2008.
P05
C1B DOMAINS OF NOVEL PKCs SHOW DIFFERENT AFFINITIES TO BIND
TO MODEL AND CELL MEMBRANES, AS A FUNCTION OF NEGATIVELY
CHARGED PHOSPHOLIPIDS AND DIACYLGLYCEROLS
Juan Carmelo Gómez-Fernández, Antonio L. Egea-Jiménez and
Senena Corbalán-García
Departamento de Bioquímica, Facultad de Veterinaria, Universidad de Murcia
The C1 domains of novel PKCs mediate the diacylglycerol-dependent translocation of
these enzymes. The four different C1B domains of novel PKCs (  and  were
studied, together with different lipid mixtures containing acidic phospholipids and
diacylglycerol or phorbol ester. The results show that either in the presence or in the
absence of diacylglycerol, C1B and C1B exhibit a substantially higher propensity to
bind to vesicles containing negatively charged phospholipids than C1B and C1B. The
observed differences between the C1B domains of novel PKCs (in two groups of two
each) were also evident in RBL-2H3 cells and it was found that, as with model
membranes, in which C1B and C1B could be translocated to membranes by the
addition of a soluble phosphatidic acid without diacylglycerol or phorbol ester, C1B and
C1B were not translocated when soluble phosphatidic acid was added, and
diacylglycerol was required to achieve a detectable binding to cell membranes. It is
concluded that two different subfamilies of novel PKCs can be established with respect
to their propensity to bind to the cell membrane and that these peculiarities in recognizing
lipids may explain why these isoenzymes are specialized in responding to different
triggering signals and bind to different cell membranes.
Acknowledgements: This work was supported by grant BFU2011-22828 from Ministerio de Economía y
Competitividad (Government of Spain) with the co-financing of the European Fund for Regional
Development (European Union) and by grant 08700/PI/08 (Fundación Séneca, Regién de Murcia, Spain).
P06
THE GP41 SEQUENCE CONNECTING MPER AND TM DOMAINS
CONSTITUTES A DISTINCT HIV-1 “FUSION PEPTIDE”
TARGETED BY NEUTRALIZING ANTIBODIES.
Beatriz Apellaniz1, Soraya Serrano2, Nerea Huarte1, Carmen Domene3, M. Ángeles Jiménez4,
José L. Nieva1.
Biochemistry & Molecular Biology, Biophysics Unit (UPV/EHU-CSIC), Leioa, Spain.
bea_apellaniz@hotmail.com
2
Institute of Physical Chemistry “Rocasolano (CSIC), Madrid, Spain,
3
Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom, 4Institute of Physical
Chemistry “Rocasolano” (CSIC), Madrid, Spain.
1
The HIV-1 virus makes use of a membrane fusion strategy to enter into the host
cell. As such, this process constitutes a clinical target for inhibitor and preventive vaccine
development. Structural, biochemical and biophysical determinations suggest that the
membrane-proximal external region (MPER) of glycoprotein 41 may interact with the
HIV-1 membrane interface, and induce its destabilization to ensue viral fusion. However,
the cholesterol content of the viral membrane (ca. 45 mol %) acts against MPER binding
and restructuring activity. Here, using vesicle stability assays, molecular dynamics
simulations, atomic force microscopy and NMR structure resolution, we have found that
gp41’s capacity for destabilizing and provoke merging of the highly rigid viral envelope,
actually resides within a sequence connecting the carboxy-terminal MPER section with
the N-terminal residues of the transmembrane domain. To determine the potential
relevance of this connection as a target for anti-HIV-1 immunogen development, we have
sought to generate antibodies against this region by immunizing rabbits with liposomepeptide formulations.
P07
FUNCTIONAL NANOSTRUCTURES BY
DESIGNED PROTEIN SELF-ASSEMBLY
Sara H. Mejías1,2, Pierre Couleaud1,2, Javier López1,3, Begoña Sot1,2, Carmen Atienza1,3,
Teresa González1, Aitziber L. Cortajarena1,2
Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia)
sara.hernandez@imdea.org
2
CNB-CSIC-IMDEA Nanociencia Associated Unit "Unidad de Nanobiotecnología"
3
Departamento de Química Orgánica, Facultad de C.C. Químicas, Ciudad Universitaria s/n,
Madrid, Spain.
1
The development of application-oriented innovative materials requires methods for control of structures
along different size scales. Bottom-up self-assembly that relies on highly specific biomolecular interactions
of small defined components, is an attractive approach for biomaterial design and nanostructure
templating1.
In this work, we used modular designed consensus tetratricopeptide repeat proteins (CTPRs) (Figura1A)2
for the generation of 3D structures (protein nanotubes) and functional nanostructures. CTPR arrays contain
multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelix
(Figure 1B)3,4.
We present the design and characterization of 3D protein-structures to use them as templates for the
creation of functional materials. We show the conjugation and characterization of the protein with gold
nanoparticles (Au-Nps) and preliminary results of the conjugation of proteins with electroactive organic
molecules.
1. The formation of the nanotubes is achieved by introducing two single point mutations in the protein to
create a new hydrophobic interface in the CTPR superhelix that will interact with identical interface on
another CTPR molecule (Figure 1C). We show the formation of protein nanotubes and characterization of
the new 3D structures.
2. The conjugation of gold nanoparticles to CTPR protein templates aims for the organization of
nanometric objects with atomic precision. Specific amino acids for metal coordination and nanoparticle
binding are introduced at defined positions of the CTPR repeat sequence. We use high resolution imaging
techniques such as atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning
transmission microscopy (STM) to characterize the CTPR-AuNps conjugates. (Figure1D).
3. We propose to use CTPR proteins in order to template donor-acceptor pairs for electroactive materials.
In order to achieve an efficient electron transfer the arrays of molecules need to be ordered with defined
inter-molecular distances. We show preliminary conjugation data in which the potential of CTPR protein
scaffolds for nanometer-precise arrangement of the molecules is explored (Figure 1E).
References
1) T. Z. Grove, L. Regan and A. L.
Cortajarena, J. R. Soc. Interface., 2013,
10, 20130051.
(2) Kajander, T.; Cortajarena, A. L.;
Regan, L. Methods Mol. Biol. 2006, 340,
151.
(3) Kajander, T.; Cortajarena, A. L.;
Mochrie, S. G.; Regan, L. Act
Crystallographica 2007, D63, 800.
(4) Cortajarena, A. L.; Wang, J.; Regan,
L. Febs J 2010, 277, 1058.
P08
OPTIMIZING A COMBINED OPTICAL TWEEZERS-LIPIDIC BILAYER
SET UP TO STUDY Ф-29 CONNECTOR
Rebeca Bocanegra1, Lara H. Moleiro2, Francisco Monroy2, José L. Carrascosa1,3
Centro Nacional de Biotecnología,CSIC, c/ Darwin 3, Cantoblanco, 28049 Madrid, Spain.
Tel: 91 585 5347; E-mail: rbocanegra@cnb.csic.es
2
Departamento de Química Física 1, Universidad Complutense, 28040 Madrid, Spain. Tel: 91 394 4128;
3
IMDEA Nanociencia, c/Faraday 9, Cantoblanco, 28049 Madrid, Spain. Tel: 91 299 8700
1
Bacteriophage Ф-29 encapsidates its DNA in a preformed prehead using its
packaging motor, located in one unique vertex of the prehead. This packaging motor
consists of three macromolecular components: the connector protein (a dodecamer
complex wich constitutes the channel), pRNA (an RNA pentamer with structural
function) and terminase (the ATPase wich provides the energy for packaging from
ATP hydrolysis).
We have developed an optimized reconstitution method for efficient orthogonal
integration of native viral connector into lipid bilayers, particularly of giant unilamelar
vesicles, made from engineered liposomes by the electroswelling method [1]. We have
optimized the bilayer in order lo afford the assembly of the complete Ф-29 motor and
we are currently optimizing the DNA packaging into liposomes with integrated
connectors (Fig.1a).
We also propose a new experimental set up based in the combination of two
powerful techniques: electrophisiology of lipid bilayers [2] and optical tweezers [3].
We are currently optimizing the isolation of a bilayer through two different
approaches, patch clamp [2] and formation of a planar lipid bilayer (PLB) [4]. With
this set up we will be able to study the forces implied in the DNA translocation through
the channel, by isolating a membrane patch with inserted connectors (Fig. 1b).
Figure 1. (a) CryoEM image of an engineered proteoliposome with an inserted Ф -29 connector in
the bilayer. (b) Schematic experimental pathway proposed. A GUV with inserted connectors in the
bilayer (red arrow) is taken to the tip of a micropipette. Appliying suction and an potential pulse we
can obtain an isolated membrane patch with one (or more) inserted connectors. We can then study
the dsDNA translocation throgh the connector pore using the optical tweezers.
Notes and References
1 Moleiro, L.H.; López-Montero, I.; Márquez, I.; Moreno, S.; Vélez, M.; Carrascosa, J.L.; Monroy,
F.ACS Synth. 2012, 1(9), 414-424
2 Molleman, A. 2003, ISBN: 0-471-48685-X
3 Hormeño, S.; Arias-Gonzalez, J.R. Biol. Cell. 2006, 98, 679-695
4 Takeuchi, S. Advances in Planar Lipid Bilayers and Liposomes 2010 11, 87-100
P09
IDENTIFICATION OF THE TRANSLOCATION STEP OF
A REPLICATIVE DNA POLYMERASE
José A. Morin1, Francisco Cao2, José M. Valpuesta3,
José L. Carrascosa3, Margarita Salas4, Borja Ibarra1
IMDEA Nanociencia, Campus Cantoblanco, 28049, Madrid, Spain
2
Universidad Complutense de Madrid, 28040, Madrid, Spain
3
Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049, Madrid, Spain
4
Centro de Biología Molecular ‘Severo Ochoa’ (CBM-UAM), Cantoblanco, 28049, Madrid, Spain
1
Replicative DNA polymerases are molecular motors that catalyze template-directed DNA
replication. In each catalytic cycle, these enzymes incorporate the correct nucleotide into
the primer or growing strand releasing pyrophosphate as a product. As a result of this
reaction replicative polymerases translocate along their DNA substrates in steps of one
nucleotide at a time (0.34 nm). Although accurate translocation is essential for genome
integrity little is known about the kinetics, energetics and integration of this process in
the nucleotide addition cycle during processive DNA replication. To address these
subjects we have used optical tweezers to manipulate individual Phi29 DNA polymeraseDNA complexes and measure the effect of mechanical force aiding and opposing
translocation on the polymerase activity at varying nucleotide (dNTPs) concentrations. A
diagram of the experimental design is shown below. Application of controlled forces on
a single polymerase biases the rates of chemical reactions involving translocation and
provided quantitative information about the ‘real time’ kinetics of elongation and the
conversion of chemical energy to motion (mechano-chemistry) during protein activity.
Fits to the replication velocity dependencies on force and dNTP concentration were
inconsistent with a model for movement incorporating a power stroke tightly coupled to
pyrophosphate release. Instead, our data is consistent with a Brownian ratchet model in
which the polymerase oscillates between the pre- and post-translocation states separated
by ~0.34 nm. The post-translocation state is energetically favored only by 0.7 KBT but it
is further stabilized by the binding of the correct dNTP.
P10
MEMBRANE PROPERTIES OF NOVEL 1-DEOXYCERAMIDES
Noemi Jiménez-Rojoa*, Aritz B. García-Arribasa, Walt A. Shawb, Jingjing Duanc,
Alfred H. Merrill, Jr. c, Félix M. Goñia, Alicia Alonsoa
a
Unidad de Biofísica (Centro Mixto CSIC, UPV/EHU), and Departamento de Bioquímica y
Biología Molecular, Universidad del País Vasco (UPV/EHU).
b
Avanti Polar Lipids, Inc. Alabaster, AL 35214 U.S.A.
c
School of Biology and Petite Institute for Bioengineering and Bioscience,
Georgia Institute of Technology, Atlanta, Georgia, U.S.A.
Serine palmitoyl transferase (SPT) is the enzyme that catalyses the first and rate limiting
step of the de novo synthesis of sphingolipids which consists of the condensation of serine
with palmitoy-CoA to produce 3-ketosphinganine. However it has been recently
demonstrated that this enzyme can also condense the non-canonical aminoacids alanine
and glycine to give rise to the novel 1-deoxysphingolipids, until present two
underappreciated lipid species. Previous data have suggested that the non-canonical 1deoxysphingolipids are biologically active as a dysregulation of their production results
in the human disease hereditary sensory autonomic neuropathy type 1 (HSAN1) (1). At
present there is no data available about the biophysical properties of these novel noncanonical 1-deoxysphingolipids. The complex biophysical properties observed in this
work suggest that these lipids may have dramatic effects in the structure and integrity of
biological membranes, particularly the endoplasmic reticulum membrane where they are
synthesized, leading, as a consequence, to ER stress. This may implicate these lipids in
important processes for cell physiology such as autophagy-mediated cell death, closely
related to ER dysfunction. Thus, their complex physico-chemical properties could be at
the origin of their pathological implications and this study opens a number of possibilities
for a function at the membrane level for these novel 1-deoxysphingolipids.
In the present contribution, different approaches have been applied to study the behaviour
of some deoxy (1-dcer), dihydrodeoxy (1-ddcer) and dihydrodeoxymethyl-ceramides (1ddmetcer). The thermotropic behavior of pure 1-deoxyceramides and of SMdeoxyceramide mixtures is described, together with the SM-deoxyceramide interactions
in monolayers, as well as the behaviour of these lipids in more complex lipid mixtures.
Acknowledgments. This work has been supported in part by grants from the Spanish
Ministerio de Economía (BFU 2012-36241; BFU 2011-28566) and the Basque
Government (IT838-13; IT849-13).
Reference
[1] Penno A, Reilly MM, Houlden H, Laura M, Rentsch K, Niederkofler V, Stoeckli ET, Nicholson G,
Eichler F, Brown RH, Jr., von Eckardstein A, Hornemann T. Hereditary sensory neuropathy type 1 is caused
by the accumulation of two neurotoxic sphingolipids. J Biol Chem. (2010) 285: 11178-11187.
P11
QUANTITATIVE DISSECTION OF STAGES IN MEMBRANE FISSION
MEDIATED BY DYNAMIN I
Eva Rodríguez Hortelano1,2, Anna Shnirova1,2, Sandra L. Smith3,
Juha-Peka Mattila3, Vadim Frolov1,2,4
2
1
Unidad de Biofisica (Centro Mixto CSIC-UPV/EHU), Leioa 48940, Spain
Departamento de Bioquímica y Biología Molecular, Universidad del País Vasco, Leioa 48940, Spain
3
Department of Cell Biology, UT Southwestern Medical Center,
5323 Harry Hines Boulevard, Dallas, TX 75390
4
IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain
Correspondence: eva.rodriguez.hortelano@gmail.com
Dynamin 1 is a key component of the protein machinery orchestrating endocytosis in
neurons, as it governs fission of the membrane neck connecting the endocytic vesicle
and the plasma membrane. Dynamin is a GTPase that converts the energy of GTP into
mechanical work of the neck constriction. This energy transduction requires
polymerization of dynamin into short helical collars enclosing the neck and
progressively tightening their grip with GTP hydrolysis. Amazingly, the neck
constriction is sufficient to trigger a complex (and irreversible) process of lipid
reorganization resulting in non-leaky membrane fission. In order to understand the
mechanism of this complex and fast process we have recently developed a method
resolving individual fission events in real time. In this study we combined this method
with specific modification of the protein (mutations interfering with its self-assembling
ability, GTP hydrolysis and consequent conformation changes) and characterized
various intermediate stages at which the fission reaction is arrested by such
modifications. By comparative analysis of these datasets we determined how the energy
is delivered from dynamin structures to the lipid matrix in the process of GTP
hydrolysis.
P12
RESIDUES IN THE TRANSMEMBRANE DOMAINS OF E1 AND E2
ENVELOPE PROTEINS OF HEPATITIS C VIRUS (HCV) INVOLVED IN
ENTRY IN Huh7 CELLS
Laura Lombana, Belén Yélamos, Julián Gómez-Gutiérrez and Francisco Gavilanes
Department of Biochemistry and Molecular Biology I, Chemistry Faculty, UCM, Madrid, Spain.
Contact: lombana.lau@gmail.com
1
Hepatitis C (HCV) virus is the major cause of acute hepatitis and chronic liver disease,
including cirrhosis and liver cancer. At this moment, there is not a vaccine available
against HCV infection and the current therapies are very expensive for undeveloped
countries [1].
The HCV envelope glycoproteins, (gp31, aminoacids192-383) and E2 (gp70, amino acids
384-746) are type-I transmembrane proteins which must play an essential role in the
initial steps of the viral infective process. The carboxy terminal transmembrane segments
(TMDs) of both envelope proteins (amino acids 341-383 for E1 and 715-746 for E2),
composed of two segments of hydrophobic amino acids separated by a short segment
containing charged residues, are responsible for attaching these proteins to the membrane
leading to their correct orientation to the outside face of the virus Thus, TMDs have been
shown to play multiple functions in the formation of the viral envelope and therefore in
the biogenesis of the HCV [2].
In order to perform a detailed characterization of these transmembrane domains we have
designed several mutants throughout these regions. We have used the system of
pseudoparticles of HCV (HCVpp), generated in HEK293T cells of human origin. The
HCVpp consist of glycoproteins of the complete envelope proteins of the HCV, E1 and
E2, assembled into retroviral particles containing a genome derived from a retrovirus
associated with a marker gene, in our case the luciferase gene [3]. These HCVpp allow
us to investigate all functions mediated by the glycoproteins of the HCV, so that we are
able to analyze the involvement of each of the mutated amino acids in integration in the
membrane and the properties of interaction and E1E2 entry into the host cell. The results
obtained using this system indicate the importance of the residues W368, K370, D728
and R730 in the TMD of E1 and E2 in the ability of the HCVpp to enter the hepatic cells
Huh7.
References:
1.
2.
3.
Ronn, R. and A. Sandstrom, New developments in the discovery of agents to treat hepatitis C.
Curr. Top. Med. Chem., 2008. 8(7): p. 533-62.
Sandrin, V., et al., Assembly of functional hepatitis C virus glycoproteins on infectious
pseudoparticles occurs intracellularly and requires concomitant incorporation of E1 and E2
glycoproteins. J Gen Virol, 2005. 86(Pt 12): p. 3189-99.
Bartosch, B., J. Dubuisson, and F.L. Cosset, Infectious hepatitis C virus pseudo-particles
containing functional E1-E2 envelope protein complexes. J Exp Med, 2003. 197(5): p. 633-42.
P13
VENEZUELAN EQUINE ENCEPHALITIS VIRUS NSP1:
MECHANISM OF ACTION OF A VIRAL CAPPING ENZYME
Jaime Guillén, Changqing Li, Julie Lichière, Bruno Canard,
Etienne Decroly and Bruno Coutard
CNRS and Aix Marseille Université, UMR7257,
Architecture et Fonction des Macromolécules Biologiques, Marseille, France
Venezuelan equine encephalitis virus (VEEV) is an infectious pathogen belonging to the
New World alphaviruses. Many of these alphaviruses are important human pathogens.
Their genomic and sub-genomic RNA are protected by a 5’ end cap structure, which is
essential for the translation of viral proteins. The nonstructural protein 1 (nsP1) is the
main enzyme for RNA capping in alphavirus. nsP1 catalyses the methylation at position
N7 of a GTP molecule and then forms a covalent link with the N7 methylated GMP,
releasing PPi. It is then supposed that this N7 Methyl GMP is transferred on the viral
mRNA. Both Methyltransferase (MTase) and Guanylyltransferase (GTase) activities of
VEEV nsP1 were characterized by means of original assays in order to uncouple both
reactions. Critical amino acids positions for both MTase and GTase functions were next
identified by site directed mutagenesis. In parallel, the binding of various RNAs and NTP
on nsP1 were characterized by biophysical methods to strengthen biochemical data.
Finally, these methods were used to evaluate the inhibition of guanylyltransferase activity
by small molecules.
Acknowledgements: This work has been sponsored by IEF Marie Curie fellowship, EUVIRNA (Marie
Curie Initial Training Network) and by the project SILVER (Health-F3-2010-260644) of the
European Union 7th Framework Program.
P14
A COMMON LINK FOR PROGRAMMED CELL DEATH
IN HUMANS AND PLANTS
Irene Díaz-Moreno, Jonathan Martínez-Fábregas, Katiuska González-Arzola,
Antonio Díaz-Quintana and Miguel A. De la Rosa
Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartuja, Universidad de Sevilla – CSIC, Sevilla, Spain
idiazmoreno@us.es
Programmed cell death (PCD) is a fundamental event for the development of
multicellular organisms. In mammalian cells, early events in PCD involve the release of
cytochrome c (Cc) from mitochondria to the cytoplasm to act at the first stages of the
apoptotic process, playing a key role in assembling the apoptosome. In plants, PCD is
part of a general process named hypersensitive response, where Cc is also released into
the cytosol but its role in PCD remains veiled. Such highly conserved cytoplasmic
location of Cc upon apoptotic stimuli lead to think of a common link for PCD in
evolutionarily distant species, like humans and plants.
To better understand the role of Cc in the onset of PCD in both humans and plants, a
proteomic approach based on affinity chromatography with Cc as bait was used. Upon
combining this approach and Bimolecular Fluorescence Complementation (BIFC), a
total of 8 human and 9 plant new proteins interacting with Cc under PCD were found
[1,2]. These new PCD Cc-partners are involved in protein folding, translational
regulation, oxidative stress, DNA damage, energetic and mRNA metabolism.
Strikingly, some of the novel human Cc-targets are closely related to those for plant Cc,
indicating that the evolutionarily well-conserved cytosolic Cc – appearing in organism
from plant to mammals – interact with a wide range of targets on PCD.
Modeling of the complexes between human and plant Cc with its counterparts shows
how the heme crevice of Cc takes part of the complex interface in agreement with the
vast majority of known redox adducts of Cc. However, in contrast to the high turnover
rate of the mitochondrial Cc redox adducts, those occurring under PCD lead to the
formation of rather stable nucleo-cytoplasmic ensembles, as inferred from Surface
Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) measurements. On
the basis of these findings, we suggest that human and plant Cc interacts with prosurvival, anti-apoptotic proteins after its release into the cytoplasm. Then, Cc may
interfere with cell survival pathways and unlock PCD in order to prevent the spatial and
temporal co-existence of antagonist signals.
Acknowledgements: This work has been sponsored by the Spanish Ministry of Economy and
Competitiveness (BFU2012-31670) and the Regional Government of Andalusia (BIO198).
References
Bernhardt R, Díaz-Quintana A, De la Rosa MA, “New Arabidopsis thaliana cytochrome c partners:
a look into the elusive role of cytochrome c in programmed cell death in plants”, Mol. Cell.
Proteomics, 12 (12), 3666-3676, 2013.
Bernhardt R, Velázquez-Campoy A, Díaz-Quintana A, De la Rosa MA, “Structural and functional
analysis of novel human cytochrome c targets in apoptosis”, Mol. Cell. Proteomics,
doi:10.1074/mcp.M113.034322, 2014.
P15
IN SILICO DESING OF NEW THERAPEUTIC MOLECULES AGAINST
DIMERIZATION OF HUMAN IGFR-1 AND EGFR RECEPTORS
José A. Encinar1, Pilar Garcia-Morales1, Gregorio J. Fernández-Ballester1,
Vicente Galiano Ibarra2, and Miguel Saceda1
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain.
Email: jant.encinar@umh.es
2
Departamento de Física y Arquitectura de Computadores, Universidad Miguel Hernández, Elche, Spain.
1
The receptor tyrosine kinases (RTKs) are a large family of plasma membrane receptors
with intrinsic protein kinase activity that transduce extracellular signals. They have a
ligand-binding domain on the extracellular face of the plasma membrane and an enzyme
active site on the cytoplasmic face, connected by a single transmembrane segment. The
cytoplasmic domain is a protein tyrosine kinase enzyme that initiates a cascade of
protein phosphorylation reactions. The receptors for insulin-like growth factor 1 (IGFR1) and epidermal growth factor (EGFR) are prototypes for this group of receptors.
Signaling through RTKs begins when the binding of agonist (i.e. IGF, EGF, etc) of
extracellular domain induce dimerization, which is supposed to bring the two
cytoplasmic tyrosine kinase domains of the receptors close enough for
autophosphorylation and to thereby activate the intrinsic tyrosine kinase activity that
triggers numerous downstream signaling pathways [1]. IGFR and EGFR are involved in
the regulation of cellular differentiation and proliferation, and are highly expressed by
many tumor cells [2, 3]. We have used the abundant structural information about these
RTKs deposited in the PDB to design peptide inhibitors of dimerization. The
reconstruction of the dimers of these receptors allows us to define protein-protein
interactions domains as potential drug targets. Using computational techniques [46] we
have designed high-affinity ligands to interfere the dimerization process. Preliminary
results show the effectiveness of different peptides in cultured cells. Peptides have been
tested in pancreatic as well as colon carcinoma cell lines where the EGFR family
members’ levels of expression are well characterized. Peptides effects on MTT
proliferation assays and cell cycle, as well as EGFR family members’ phosphorylation
are under way with promising results.
Acknowledgements: This work has been sponsored by FIS PI012/02025,
References
[1] H. Ogiso, R. Ishitani, Nureki O, Fukai S, Yamanaka M, Kim JH, Saito K, Sakamoto A, Inoue M,
Shirouzu M, Yokoyama S. “Crystal structure of the complex of human epidermal growth factor and
receptor extracellular domains.”, Cell, 110, 775-787, 2002.
[2] R.I. Nicholson, J.M.W. Gee, M.E. Harper. “EGFR and cancer prognosis.”, European Journal of
Cancer, 37, 9-15, 2001.
[3] Worrall C, Nedelcu D, Serly J, Suleymanova N, Oprea I, Girnita A, Girnita L. “Novel mechanisms
of regulation of IGF-1R action: functional and therapeutic implications.”, Pediatr Endocrinol Rev.,
10, 473-484, 2013.
[4] Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. “The FoldX web server: an
online force field.”, Nucleic Acids Res., 33, W382-388, 2005.
[5] ADAN database site: http://adan-embl.ibmc.umh.es/.
[6] FoldX computer algorithm web site: http://ww.foldx.org/.
P16
TubZ FILAMENTS CONFORMATIONAL CHANGE UNDERLYING
GTP HYDROLYSIS: C TERMINAL TAIL IMPLICATION
IN THE OPENING OF THE PROTOFILAMENT TWIST
María A. Oliva1, María E. Fuentes2 and Fernando Moreno2
CSIC-Centro de Investigaciones Biológicas. c/ Ramiro de Maeztu, 9. 28040-Madrid, Spain.
marian@cib.csic.es
2
CSIC- Centro Nacional de Biotecnología. c/ Darwin, 3. 28049-Madrid, Spain
1
TubZ is the latest described member in tubulin/FtsZ superfamily of self-assembly
GTPases and is involved in plasmids and phages DNA distribution within the bacteria
[1, 2, 3]. Showing a tubulin-like polar protofilament formation, TubZ assembles into
double helical filaments [4] that self-organize and follow a treadmilling dynamics,
similarly to actin filaments [5, 6].
Considering that dynamicity is crucial for the function of cytomotive filaments, we have
studied the structural conformational change in TubZ filaments due to GTP hydrolysis.
We have used a biochemical and structural approach to understand the direction of the
changes and how they drive the disassembly process. We have found that the transition
is different from the straight-to curved change described in other tubulin-like filaments.
Instead, in TubZ filament there is an opening of the helical structure by increasing of the
inter-monomer twist (straightening of the protofilament), which becomes incompatible
with treadmilling dynamics. Further, the C-terminal tail is: i) responsible of the
formation of a functional double helical filament by getting the appropriate intermonomer twist, ii) necessary for spreading the changes induced by GTP hydrolysis
along the filaments and, iii) important in the interaction with its partner protein TubY
during the re-modeling of the filaments into FtsZ/tubulin-like rings structures.
Acknowledgements: This work has been sponsored by RyC program (2011) and Jose M. Andreu’s
grant BFU2011-23416.
References
[1] L Ni, W Xu, M Kumaraswami, MA Schumacher, Plasmid protein TubR uses a distinct mode of
HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition, Proc
Natl Acad Sci U S A, 107 (26), 11763-8, 2010.
[2] MA Oliva, AJ Martin-Galiano, Y Sakaguchi, JM Andreu, Tubulin homolog RubZ in a phageencoded partition system, Proc Natl Acad Sci U S A, 109 (20), 7711-6, 2012.
[3] JA Kraemer, ML Erb, CA Waddling, EA Montabana, EA Zher, H Wang, K Nguyen, DS Pham, DA
Agard, J Pogliano, A phage tubulin assembles dynamic filaments by an atypical mechanism to center
viral DNA within the host cell, Cell, 149 (7), 1488-99, 2012.
[4] CH Aylett, T Izore, LA Amos, J Löwe, Filament structure of bacterial tubulin homologue TubZ,
Proc Natl Acad Sci U S A, 107 (46), 19766-71, 2010.
[5] Y Chen, HP Erickson, In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus
plasmids: evidence for a capping mechanism, J Biol Chem, 283 (13), 8102-9, 2008.
[6] RA Larsen, C Cusumano, A Fujioka, G Lim-Fong, P Patterson, J Pogliano, Treadmilling of a
prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis,
Genes Dev, 21 (11), 1340-52, 2006.
P17
MECHANICAL STABILITY OF THE NATURAL DnaJ ZN-FINGER DOMAIN
REVEALED BY SINGLE MOLECULE FORCE SPECTROSCOPY
Judit Perales-Calvo, Ainhoa Lezamiz and Sergi Garcia-Manyes
Department of Physics and Randall Division of Cell and Molecular Biophysics,
King’s College London, Strand, London WC2R 2LS
Zinc is an abundant metal essential for life that is present in ca.2800 proteins in the human
proteome. When coordinated inside a protein structure it often forms a stable structural
motif, the so-called zinc finger [1]. DnaJ is a 5-domain Hsp40 molecular chaperone that
naturally binds two atoms of Zn2+, each one coordinated to two –CXXC- chelating motifs,
acquiring an unusual tetrahedral C4-type Zn-finger topology [2]. Here we use single
molecule force spectroscopy AFM to unravel the mechanical stability of each
independent DnaJ zinc finger. Upon pulling the protein at a constant velocity, we first
discover that a 3-domain mutant of DnaJ (DnaJ107) follows a sequential unfolding
mechanism that does not follow a common mechanical hierarchy scenario. On a typical
unfolding trajectory, the mechanically labile domain III unfolds first, requiring a force of
105  44 pN, followed by the unfolding of domain I, occurring at 191  39 pN. Crucially,
the hidden Zn binding domain is exposed to the mechanical force only when domain I
has already unfolded. The force required to break an individual Zn-S bond, triggering the
unfolding of both Zn fingers, is surprisingly low, of 90  11pN. Pulling on an Apo DnaJ
mutant (DnaJ107(C161/164/197/200S)), whereby two cysteines are mutated in each finger to
impair Zn2+ binding, reveals that in half of the trajectories the apo-form (devoid of
mechanical stability) is formed. However, in the other half of the trajectories, a single
new ZnS4 centre that features a mechanical stability akin to that of both native fingers is
unexpectedly observed, demonstrating the plasticity of the structure as both Zn-fingers in
DnaJ are able to hybridize. Our results provide direct quantification of the mechanical
role of individual Zn-thiolate bonds, of widespread presence in nature.
[1] Berg, JM. and Shi, Y. (1996). Science. 271: 1081-5.
[2] Martinez-Yamout, M., Legge, GB., Zhang, O., Wright, PE. and Dyson, HJ. (2000). J Mol Biol. 300:
805-18.
P18
CELL WALL REMODELLING BY THE ZINC-PROTEASES AMPDH2 AND
AMPDH3, VIRULENCE DETERMINANT OF PSEUDOMONAS
AERUGINOSA, AND LYTIC TRANSGLYCOSYLASE MLTC.
Cecilia Artola-Recolons1, Mijoon Lee2, Siseth Martínez-Caballero1, Noelia Bernardo-García1,
Sergio G. Bartual1, César Carrasco-López1, Blas Blázquez2, Dusan Hesek2, Edward Spink2,
Elena Lastochkin2, Weilie Zhang2, Lance M. Hellman2, Bill Boggess2, Kiran V. Mahasenan2,
Kathrin Meindl3, Isabel Usón3, Shahriar Mobashery2 and Juan A.Hermoso1.
Instituto de Química-Física “Rocasolano”. Departamento de Cristalografía y Biología Estructural. CSIC,
Serrano 119, 28006 Madrid, Spain.
2
University of Notre Dame. Department of Chemistry and Biochemistry,
Nieuwland Science Hall, Notre Dame, Indiana 46556, USA
3
Instituto de Biología Molecular de Barcelona, CSIC, Institucio Catalana de Recerca y Estudis Avançats,
Baldiri Reixach 13, 08028 Barcelona, Spain
1
Bacterial cell wall is a polymer of considerable complexity that is in constant
equilibrium between synthesis and recycling. The periplasmic zinc proteases AmpDh2
and AmpDh3 are a virulence determinant of Pseudomonas aeruginosa, a problematic
human pathogen. The mechanism of how the protease manifests virulence is not known,
though it is related to the turnover of the bacterial cell wall. In this work we document
the reactions that these enzymes perform on the cell wall, hydrolyzing the peptide stems
from the peptidoglycan. Based on the X-ray structures of both enzymes in complex with
synthetic cell-wall-based ligands, we present the possible mechanism of action of these
two enzymes in vivo. Besides, cell Wall recycling mechanism of bacteria also needs the
action of important enzymes called lytic transglycosylase. MltC is the head of a family
present in 791 bacterial genomes, and it has been suggested to play a key role in
bacterial physiology. The X-ray structure of MltC with three synthetic analogues has
revealed the nature of interactions of MltC with the cell wall.
Acknowledgements: This work was supported by grants, GM61629 (the US National Institutes of
Health), BFU2011-25326 (the Spanish Ministry of Economy and Competitiveness), S2010/BMD2457 (the Government of Community of Madrid), Juan de la Cierva grant, Spanish MINECO and
Generalitat de Catalunya for financial support (IDC-20101173 and 2009SGR-1036). The Mass
Spectrometry & Proteomics Facility of the University of Notre Dame is supported by grant
CHE0741793 from the US National Science Foundation
References
[1] Siseth Martínez-Caballero*, Mijoon Lee*, Cecilia Artola-Recolons*, César Carrasco-López, Dusan
Hesek, Edward Spink, Elena Lastochkin, Weilie Zhang, Lance M. Hellman, Bill Boggess, Shahriar
Mobashery, Juan A. Hermoso. “Reaction products and the X-ray structure of AmpDh2, a virulence
determinant of Pseudomonas aeruginosa”, Journal of the American Chemical Society, 135,
10318−10321, 2013.
[2] Mijoon Lee*, Cecilia Artola-Recolons*, César Carrasco-López*, Siseth Martínez-Caballero, Dusan
Hesek, Edward Spink, Elena Lastochkin, Weilie Zhang, Lance M. Hellman, Bill Boggess, Juan A.
Hermoso, Shahriar Mobashery. “Cell-Wall Remodeling by the Zinc-Protease AmpDh3 from
Pseudomonas aeruginosa” , Journal of the American Chemical Society, 135, 12604−12607, 2013.
[3] Cecilia Artola-Recolons*, Mijoon Lee*, Noelia Bernardo-García, Blas Blázquez, Dusan Hesek,
Sergio G. Bartual, Kiran V. Mahasenan, Elena Lastochkin, Hualiang Pi, Bill Boggess, Kathrin
Meindl, Isabel Usón, Jed F. Fisher, Shahriar Mobashery*, and Juan A. Hermoso. “X-Ray Structure
and Reactions for the Lytic Transglycosylase MltC of Escherichia coli”, Angewandte Chemie,
Submitted, 2014.
P19
DUALLY PREDICTED COILED-COIL AND DISORDERED REGIONS OF
CENTROSOMAL PROTEINS: EMERGENCE OF STRUCTURE THROUGH
PROTEIN-PROTEIN INTERACTIONS AND pH CHANGES
Marta Bruix1, Miguel A. Treviño1, M. Flor García-Mayoral1,
M. Angeles Jiménez1, Ugo Bastolla2
1
Instituto de Química Física “Rocasolano”, CSIC, Madrid, Spain. mbruix@iqfr.csic.es
Centro de Biología Molecular Severo Ochoa (CBMSO), CSIC-UAM, Cantoblanco, Madrid, Spain
2
Human centrosomal proteins show a significant, 3.5 fold, bias to be both unstructured
and coiled-coils based on results from state of the art bioinformatics tools. We
hypothesize that this bias means that these proteins adopt an ensemble of disordered and
partially helical conformations, with the latter becoming stabilized when these proteins
form intermolecular complexes. To test this hypothesis, and to provide experimental
data for improving bioinformatics methods, we have employed biophysical methods
(NMR, CD, Ultracentrifugation, Light Scattering) to characterize the structural
properties of 13 such peptides ranging in size from 20 to 61 residues. Our hypothesis
was confirmed in most cases. For two of the peptides, PIK3R1453-513 and BRCA112531273
, we observed structure stabilization through self-association, but also the formation
of -sheets linked to the formation of high molecular weight aggregates unobservable
by liquid state NMR, but are the predominant forms detected by CD. Four peptides,
derived from the three proteins CCNA2103-123, BRCA11253-1273, BRCA11397-1424 and
PIK3R1453-513, can form intermolecular associations that stabilize the secondary
structure, whereas the transient interactions detected for the kinase NEK2 did not lead to
any stabilization in the NEK2303-333, NEK2341-361, and NEK2410-430 peptide’s structure.
Interestingly, the secondary structure of most of these peptides becomes stabilized at
acidic pH. Our results point out the importance of combining different biophysical
techniques before deriving conclusions on the behavior of mainly disordered or partly
structured proteins. All these results, obtained from 13 different peptides derived from
10 different centrosomal proteins, allow us to advance that conformational
polymorphism, modulated by intermolecular interactions is a general property of
centrosomal proteins.
Acknowledgements: This work has been sponsored by the projects Consolider-Ingenio Centrosome3D CSD2006-00023, CAM S2010/BMD-2305, BFU2012-40020 and CTQ2011-22514.
P20
STRUCTURAL BASIS OF PcsB-MEDIATED CELL SEPARATION
IN Streptococcus pneumoniae
Sergio G. Bartual1, Daniel Straume2, Gro Anita Stamsås2, Inés G. Muñoz3, Carlos Alfonso4,
Martín Martínez-Ripoll1, Leiv Sigve Håvarstein 2 & Juan A. Hermoso1
1
Department of Crystallography and Structural Biology, Instituto de Química-Física Rocasolano,
CSIC, Serrano 119, 28006-Madrid, Spain.
Department of Chemistry, Biotechnology and Food Science,
Norwegian University of Life Sciences, N-1432, Ås, Norway.
3
Macromolecular Crystallography Group. Structural Biology and Biocomputing Programme, Spanish
National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029-Madrid, Spain.
2
4
Centro de Investigaciones Biológicas (CIB), Ramiro de Maeztu 9, 28040-Madrid, Spain.
In order to divide, bacteria must synthesize a new septal cell wall, which must be split
down the middle by one or more murein hydrolases to separate the resulting daughter
cells. In the Gram-positive bacterium Streptococcus pneumoniae, PcsB is predicted to
perform this operation. Recent evidence shows that PcsB is recruited to the septum by
the transmembrane FtsEX complex, and that this complex is required for cell division.
However, PcsB lacks detectable catalytic activity in vitro, and while it has been
proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate
that PcsB has muralytic activity, and report the crystal structure of full-length PcsB [1].
The protein adopts a dimeric structure in which the V-shaped coiled-coil domain of
each monomer acts as a pair of molecular tweezers locking the catalytic domain of each
dimeric partner in an inactive configuration. This suggests that the release of the
catalytic domains likely requires an ATP-driven conformational change in the FtsEX
complex, conveyed towards the catalytic domains through coordinated movements of
the coiled-coil domain.
Acknowledgements: This work was supported by grants BFU2011-25326 and S2010/BMD-2457 and
grants from the Research Council of Norway.
[1] Sergio G. Bartual, Daniel Straume, Gro Anita Stamsås, Inés G. Muñoz, Carlos Alfonso, Martín
Martínez-Ripoll, Leiv Sigve Håvarstein & Juan A. Hermoso. “Structural basis of PcsB-mediated cell
separation in Streptococcus pneumoniae” Nature Communications. (2014) In press.
doi:10.1038/ncomms4842
P21
EQUILIBRUM AND DYNAMIC INTERFACIAL PROPERTIES OF
PULMONARY SURFACTANT STUDIED BY NEW MICROPIPETTE
TECHNIQUES
Elisa Parra1, Koji Kinoshita1, Jesús Pérez-Gil2, David Needham1,3
Center for Single Particle Science and Engineering (SPSE), Syddansk Universitet, Odense, Denmark.
2
Dep. Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain.
3
Dep. Mechanical Engineering and Material Science, Duke University, Durham, NC, USA.
1
The respiratory surface of mammalian lungs is stabilized by pulmonary surfactant, a
membrane-based system composed by multiple lipids and specific proteins whose main
function is minimizing the surface tension at the alveolar air-water interface. Deficiencies
or alterations in this system are directly related to several respiratory pathologies
including neonatal and acute respiratory distress syndromes or meconium aspiration
syndrome in newborns. It is therefore crucial to understand the fundamental properties of
natural lung and clinical surfactants in order to improve surfactant therapies. The overall
goal of the present work is to establish the micropipette technique as a novel method for
the evaluation of lung surfactants, both natural and clinical, in terms of surface activity
and mechanical properties at air-water interfaces.
In order to achieve this, synthetic lipid mixtures, clinical formulations and native
surfactant from porcine lungs are studied and compared. A microscopic interface (R  10
– 100 m) is formed inside a tapered micropipette. Lipid and surfactant-coated interfaces
are characterized in terms of equilibrium surface tensions, by measuring the radius of
curvature of the interface inside the pipette for a series of applied pressures [1,2].
Dynamic surface tensions are recorded upon the adsorption of new material to a clean airwater interface. Phospholipid-cholesterol mixtures and the commercial product Infasurf®
(calf lung surfactant) reduce the surface tension of an air-water interface at 37C from 70
mN/m to equilibrium values around 20-21 mN/m in less than 60 s. We have also
developed a new method using a trumpet-shaped pipette in which the surface-active
materials are added from the air side onto the air-water interface using a delivery pipette,
which aims to reproduce surfactant spreading from the airways.
The versatility of this technique is being further explored by characterizing gas
microbubbles coated with the different lipid mixtures and surfactants, modeling artificial
alveoli. They can be used to measure gas dissolution into the aqueous medium as a
function of the surface tension, which in turn creates a Laplace overpressure inside the
lipid-coated bubble [3]. They can also be manipulated to obtain viscoelastic properties of
the interfacial films, such as yield shear and shear viscosity [4]. The studies presented
here will improve our comprehension of lung surfactant as well as provide a new and
versatile platform for surfactant evaluation and regulatory approval of clinical products.
References:
[1] Lee, S., D.H. Kim, and D. Needham. Langmuir, 2001. 17(18): p. 5544-5550.
[2] Lee, S., D.H. Kim, and D. Needham. Langmuir, 2001. 17(18): p. 5537-5543.
[3] Duncan, P.B., and D. Needham. Langmuir, 2004. 20(7): p. 2567-2578.
[4] Kim, D.H., et al. Langmuir, 2003. 19(20): p. 8455-8466.
P22
MOLECULAR DETERMINANTS IN THE Kv1.3 C-TERMINUS ARE
RESPONSIBLE FOR CHANNEL ANTEROGRADE TRANSPORT AND
SURFACE EXPRESSION VIA COPII-DEPENDENT MECHANISMS
Ramón Martínez-Mármol1, Mireia Pérez-Verdaguer1, Sara R. Roig1, Albert Vallejo-Gracia1,
Jesusa Capera1, Antonio Serrano-Albarrás1, Clara Serrano-Novillo1, Antonio Ferrer-Montiel2,
Gregorio Fernández-Ballester2, Núria Comes1, Antonio Felipe1
Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain,
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain.
1
2
Impairment of Kv1.3 membrane expression in leukocytes and sensory neuron
contributes to the pathophysiology of autoimmune diseases and sensory syndromes.
Molecular mechanisms underlying Kv1.3 channel trafficking to the plasma membrane
remain elusive. We report a novel non-canonical di-acidic signal (E483/484) at the Cterminus of Kv1.3 essential for anterograde transport and surface expression. Notably,
homologous motifs are conserved in neuronal Kv1 and Shaker channels. Biochemical
analysis reveals interactions with the Sec24 subunit of the coat protein complex II.
Disruption of this complex drastically retains the channel at the endoplasmic reticulum.
A molecular model of the Kv1.3-Sec24a complex suggests salt-bridges between the diacidic E483/484 motif in Kv1.3 and the di-basic R750/752 sequence in Sec24. These
findings identify a novel and previously unrecognized motif of Kv channels essential
for their expression in the cell surface. Our results contribute to our understanding of
how Kv1 channels target to the cell membrane, and provide new therapeutic strategies
for the treatment of pathological conditions
Acknowledgements: Supported by BFU2011-23268 and CSD2008-00005 from the MINECO, Spain
P23
PHYSIOLOGICAL ROLE OF THE COLLABORATION OF Kv1.3
WITH CAVEOLIN DURING ADIPOGENESIS
Mireia Pérez-Verdaguer1,2, Jesusa Capera1,2, Clara Serrano-Novillo1,2, Joanna Bielańska1,2,
Marta Camps2, Anna Gumà2, Núria Comes1,2 and Antonio Felipe1,2
Molecular Physiology Laboratory, 2Departament de Bioquímica i Biologia Molecular,
Institut de Biomedicina (IBUB), Universitat de Barcelona. (Spain)
1
The voltage-dependent K+ channel Kv1.3 is involved in a myriad of physiological
events in leukocytes, sensory neurons, vascular smooth muscle and adipocytes. Kv1.3
concentrates in lipid rafts. These domains are signal platforms where signalling
molecules and targets converge. Specialized forms of rafts are caveolae. These omega
shaped structures are highly abundant in adipocytes where they account for 30% of the
plasma membrane surface. Their structure is due to the presence of caveolin 1 which
participates in the transport of cholesterol from endoplasmic reticulum to plasma
membrane.
The role of Kv1.3 in adipocytes raises an important debate as it has been proposed that
Kv1.3 could be a pharmacological target in obesity. Because the localization of the
channel is important for its function, in the present work we studied the presence and
localization of Kv1.3 in adipocytes. We have characterized the presence of Kv1.3 in rat
and human adipocytes and during adipogenesis of the 3T3-L1 cell line. Adipogenesis
leads to caveolin 1 (Cav1) expression and the appearance of caveolae; therefore, we
analyzed the microdomian localization of Kv1.3. In addition, by lentiviral infection, we
generated different 3T3-L1 cell lines genetically deficient in caveolin 1 and Kv1.3, in
order to study any putative phenotypically reprograming. We found that in Cav1-null
cells, Kv1.3 shifted floatability. In addition, Kv1.3-null cells did not reach adipocyte
differentiation because an impaired proliferative behaviour. Our results indicate that
adipogenesis triggers a relocalization of Kv1.3 in newly synthesized caveolae. Further,
Kv1.3 is crucial for the regular cell cycle progression in 3T3-L1 pre-adipocytes.
Because most of insulin-dependent adipocyte signalling machinery is located in these
structures, our results bring light to the role of Kv1.3 in the adipocyte physiology.
Supported by BFU2011-23268 and CSD2008-00005 from the MINECO, Spain
P24
CHARACTERIZATION OF A NEW FAMILY OF BROAD-SPECTRUM
RACEMASES INVOLVED IN PRODUCTION OF NONCANONICAL
D-AMINO ACIDS AND CELL-WALL REGULATION
Noelia Bernardo-García1, Cesar Carrasco-López1, Akbar Espaillat2,4, Natalia Pietrosemoli3,
Lisandro H. Otero1, Laura Álvarez2,4, Miguel A. de Pedro4, Florencio Pazos3, Brigid M. Davis5,
Mathew K. Waldor5, Felipe Cava2,4 and Juan A. Hermoso1
Department of Crystallography and Structural Biology, Instituto de Química-Física ‘Rocasolano’–CSIC,
28006 Madrid, Spain.
Email: xnoelia@iqfr.csic
2
Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden,
Umeå Centre for Microbial Research, Umeå, University, Umeå, Sweden.
3
Centro Nacional de Biotecnología–CSIC, 28049 Madrid, Spain.
4
Centro de Biología Molecular ‘Severo Ochoa’, Universidad Autónoma de Madrid–Consejo Superior de
Investigaciones Científicas (CSIC), 28049 Madrid, Spain,
5
Division of Infectious Diseases, Brigham and Women’s Hospital and Department of Microbiology and
Immunobiology, Harvard Medical School and HHMI, Boston, MA 02115, USA
1
Broad-spectrum amino acid racemases (Bsrs) enable bacteria to generate noncanonical
D-amino acids (NCDAA), whose roles in microbial physiology, including modulation
of cell wall structure and dissolution of biofilms, are just beginning to be appreciated.
Here we used crystallographic, mutational, biochemical and molecular simulation
studies to define the molecular features of the racemases BsrV from Vibrio cholerae,
BsrAb from Acinetobacter baumanii and BsrKO from Kingella oralis. These enzymes
are able to accommodate more diverse substrates than related PLP-dependent alanine
racemases. We identified conserved residues that distinguish BsrV and a newly defined
family of broad-spectrum racemases from the classical alanine racemases, and found
that these residues are key mediators of BsrV's multispecificity. This new family
presents the enzymes located in the periplasm and not in the cytoplasm as the Alaracemases. Surprisingly, NCDAA-modified cell wall peptides were found to exert a
strong inhibitory effect on Bsrs activity. We propose that such modified muropeptides
underlie a negative feedback loop that prevents excessive NCDAA production and
controls the cell wall biosynthesis.
Reference
Espaillat, A., Carrasco-López, C., Bernardo-García, N., Pietrosemoli, N., Otero, L. H., Álvarez, L., de
Pedro, M. A., Pazos, F., Davis, B.M., Waldor, M.K., Hermoso, J.A., and Cava, F. “Structural basis for
the broad specificity of a new family of amino-acid racemases” Acta Crystallographica Section D
Biological Crystallography, 70, 79–90, 2014.
P25
FLUORESCENT POLYENE CERAMIDE ANALOGUES
AS MEMBRANE PROBES
Ingrid Nieves1, Ibai Artetxe2, L. Ruth Montes2,3, Jesús Sot2, José Luis Abad1, Alicia Alonso2,
Lluís Fajarí4, Antonio Delgado1,5, Félix M. Goñi2
Research Unit on Bioactive Molecules (RUBAM), Dept. of Biomedicinal Chemistry, IQAC-CSIC,
Barcelona, Spain. 2 Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica,
Universidad del País Vasco, Bilbao, Spain. 3 University of the Basque Country UPV/EHU, Dept. of
Physiology, Faculty of Pharmacy, Vitoria-Gasteiz, Spain. 4 Dept. of Biological Chemistry and Molecular
Modelling, IQAC-CSIC, Barcelona, Spain. 5 University of Barcelona, Faculty of Pharmacy, Unit of
Pharmaceutical Chemistry (Associated Unit to CSIC), Barcelona, Spain.
1
Fluorescence spectroscopy is a frequently used technique in the field of membrane
biophysics. Many different chemically modified lipids have been used as fluorescent
probes in studies on the field. However, most modifications consist on the addition of a
bulky fluorophore such as Bodipy or NBD, which can drastically disrupt the lipid
properties. In a less disturbing alternative, 4-5 conjugated double bonds can be added to
the acyl chain of the lipids [1]. The resulting molecules are fluorescent systems
uniquely similar to the natural lipids, suitable for a wide range of fluorescent studies,
including live-cell imaging. Efforts are under way to synthesize a ceramide pentaene
with 5 conjugated double bonds in its sphingoid base, which would be of great interest
for membrane sphingolipid microdomain studies. Additionally, addition of a radical
quencher in its acyl chain would allow spatio-temporal modulation of the fluorescent
emission through the action of a specific ceramidase. As a proof of concept, a series of
ceramide-resembling model GABA-pentaene fluorescent probes have been synthesized,
with an internal n-doxyl stearoyl radical as a fluorescence quencher and spin marker.
The fluorescent properties of these molecules were studied in liposomes by
spectrofluorometry, EPR and fluorescence confocal/two-photon microscopy. The
fluorescent emission of the doxylated probes was shown to depend on the position of
the doxyl group and on the lipidic environment. While emission of Penta5dox did not
change much between lipid compositions Penta16dox showed a markedly increase in
emission on fluid liposomes compared to gel ones. EPR studies explained this as a
result of differences in intramolecular fluorescence quenching due to the conformational
mobility of the n-doxyl stearoyl group. Furthermore, the Pentaene I probe was shown to
be an interesting probe for fluorescent microscopy studies on domain formation in
model membranes, due to its preferential staining of ordered domains on giant
unilamellar vesicles. In summary, these GABA-pentaene compounds may be interesting
as novel fluorescent lipid probes for studies on membrane domains. In addition, they are
also valuable models that justify the synthetic efforts required for the synthesis of the
initially proposed fluorescent ceramide probe.
Acknowledgements: This work has been sponsored by grants from the Spanish Ministry of Economy
((BFU 2012-36241 to FMG, BFU 2011-28566 to AA) and from the Basque Government (Grant Nos. IT
849-13 to FMG and IT 838-13 to AA).
References
[1] L. Kuerschner, C.S. Ejsing, K. Ekroos, A. Shevchenko, K.I. Anderson, C. Thiele, Polyene-lipids: a
new tool to image lipids, Nature Methods, 2 (2005) 39-45.
P26
TOWARDS VISUALIZING DNA REPAIR AT THE SINGLE MOLECULE
LEVEL COMBINING MAGNETIC TWEEZERS AND TIRF MICROSCOPY
Julene Madariaga Marcos1, Mark S. Dillingham2 and Fernando Moreno Herrero1
Department of Macromolecular Structures, Centro Nacional de Biotecnología, Consejo Superior de
Investigaciones Científicas, Madrid, Spain. E-mail: jmadariaga@cnb.csic.es
2
DNA:Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol, United Kingdom
1
Double-strand breaks (DSB) are a source of DNA damage frequently produced
during the normal metabolism of cells. If not properly repaired, DSB can lead to
genomic instability, developmental defects and cancer. Fortunately, cells posses robust
repair mechanisms, such as homologous recombination, which relies in using the sister
chromatid as a template to copy and repair the damaged DNA strand [1].
In bacteria, it has been precisely and extensively described how helicases and
nucleases like AddAB/RecBCD perform the first step in this repair process, unwinding
and degrading DNA, regulated by Chi recombination sequences [2-4]. We have
previously characterized the unwinding activity of the AddAB helicase-nuclease using
AFM [3] and a Magnetic Tweezers setup [5]. Now we aim to incorporate to our setup
total internal reflection fluorescence (TIRF). This is a powerful approach because it will
allow us to correlate biological activity (such as translocation) with precise positioning
and stoichiometries (directly observed by fluorescence) of the repair proteins.
We decided to implement an objective-type TIRF, where the excitation beam is
directed to the sample surface in total reflection and the fluorescence emission is
collected by the same objective, for which, high NA is necessary. This implementation
is quite straightforward, as no modification of the current magnet heads is required.
We will use a 488nm laser as excitation source, expanded 7 times and circularly
polarized for homogeneous excitation. To reach total internal reflection, we will
translate a mirror placed in a micrometric stage. In this way, combined with the
objective approach, switching for TIRF to epi-illumination will be easily achieved.
Finally, several dichroic mirrors placed on the emitted fluorescence path will allow light
from the tweezers and fluorescence to reach their corresponding detectors in a handy
way.
Acknowledgements: J.M.M. acknowledges support of the Basque Government through a Predoc
fellowship (ref PRE_2013_11_1174).
References
[1] Claire Wyman and Roland Kanaar , Annu. Rev. Genet., 40, 363-83, 2006.
[2] Piero C. Bianco and Stephen C. Kowalczykowski, Proc. Natl. Acad. Sci. USA, 94, 6706–11, 1997.
[3] Joseph T.P. Yeeles et al., Mol. Cell, 42, 806-16, 2011.
[4] Dale B. Wigley, Nat. Rev. Microbiol., 11, 9-13, 2013.
[5] Carolina Carrasco et al., Proc. Natl. Acad. Sci. USA, 110(28), E2562-71, 2013.
P27
QUANTIFYING EQUIVALENT POLARITY AND EXCIMER FORMATION IN
UNSATURATED LIPID BILAYERS: SIGNIFICANCE OF MOLECULAR
PROPERTIES OF PYRENE PROBES IN FLUORESCENCE STUDIES
Jorge Martins1,2, Dalila Arrais1 and Miguel Manuel1
IBB-CBME, Universidade do Algarve, Campus de Gambelas, P-8005-139, Faro, Portugal
DCBB-FCT, Universidade do Algarve, Campus de Gambelas, P-8005-139, Faro, Portugal
1
2
Pyrene based fluorophores present diverse advantages, e.g. high quantum yields, long
lifetimes, and precise solvatochromic effects, most suitable to probe varied biophysical
phenomena in lipid bilayer [1]. Although often used in fluorescence studies, their
molecular and photophysical properties have been frequently overlooked. Knowledge
on the physical-chemical characteristics of probes and on their location allows obtaining
correct information about the milieu sensed within lipid bilayers. The pyrene moiety
being an apolar and bulky group displays distinct locations as a free molecule [2] or
linked to phospholipid chains [3]. The location of free pyrene defines the values for the
bilayer dielectric constant being averaged transversally in space and laterally in time.
Also, the mutual hydrophobic interactions of the rigid polyaromatic hydrocarbon with
acyl chain’s methylenic groups of larger conformational freedom increasing chain order
is consenting an unused analysis of excimer formation at moderate probe proportions.
We used the Ham Effect in pyrene spectrum to monitor the bilayer polarity of pure
unsaturated POPC or DOPC and in their binary mixtures with egg-sphingomyelin (eggSM) or cholesterol (Chol). Pure DOPC exhibits higher dielectric constants than POPC,
due to larger free volume enabling elevated hydration within lipid bilayers. POPC/Chol
DOPC/Chol and DOPC/egg-SM mixtures display similar variation of bilayer polarity,
pointing to related features in their thermal phase diagrams. The bilayer polarity of
POPC/egg-SM mixture was no varying as a function of egg-SM content.
We used py10-PC and py6-PC pyrenyl-labeled phosphatidylcholines in POPC fluid
bilayers to analyze the excimer formation reaction by using steady-state and lifetime
fluorescence. We found very good agreement with the theoretical predictions of a
kinetic formalism for fluorescence self-quenching processes occurring in twodimensional (2D) media [4]. However, significant downward divergences occur in 2D
Förster-Kasper plots above 2 mol % for py10-PC and 4 mol % for py6-PC content,
revealing deviations to the assumed constancy in the probes’ lateral diffusion dynamics.
Acknowledgements: This work has been supported by national Portuguese funding through FCT Fundação para a Ciência e a Tecnologia, project ref. Pest-OE/EQB/LA0023/2012 and project ref.
PTDC/QUI-BIQ/112943/2009.
References
[1] Eurico Melo, Jorge Martins, “Kinetics of bimolecular reactions in model bilayers and biological
membranes. A critical review”, Biophys. Chem., 123, 77-94, 2006.
[2] Luís M.S. Loura, António M.T. Martins do Canto, Jorge Martins, “Sensing hydration and behavior
of pyrene in POPC and POPC/cholesterol bilayers: a molecular dynamics study”, Biochim.
Biophys. Acta - Biomembranes, 1828, 1094-1101, 2013.
[3] Jarmila Repáková, Juha M. Holopainen, Mikko Karttunen, Ilpo Vattulainen “Influence of pyrenelabeling on fluid lipid membranes”, J. Phys. Chem. B, 110, 15403-15410, 2006.
[4] K. Razi Naqvi, Jorge Martins, Eurico Melo, “Recipes for analyzing diffusion-controlled reactions in
two dimensions: time-resolved and steady-state measurements”, J. Phys. Chem. B, 104, 1203512038, 2000.
P28
NOSOLOGICAL IMAGING OF GLIOBLASTOMA RESPONSE TO THERAPY
Magdalena Ciezka1,2,3, Teresa Delgado-Goñi4, Sandra Ortega-Martorell5,2, Ivan Olier6,
Margarida Julià-Sape2,1,3, Ana Paula Candiota2,1,3, Paulo J.G. Lisboa5, Carles Arús1,2,3
Department de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Edifici Cs,
Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
2
Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina
(CIBER-BBN), Spain
3
Institut de Biotecnologia i de Biomedicina, Cerdanyola del Vallès, Spain
4
The Institute of Cancer Research, London, United Kingdom
5
Department of Mathematics and Statistics, Liverpool John Moores University,
Liverpool, United Kingdom
6
Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom
1
Introduction: Glioblastomas (GBM) are high-grade brain tumours with poor survival
and bad prognosis. There are no robust methods for early assessment of therapy
response available. The rich information contained in magnetic resonance (MR) signals
(i.e. spectroscopy, MRS/ spectroscopic imaging, MRSI) makes them ideally suited for
the application of pattern recognition (PR) techniques. Source extraction analysis can
provide the identification of tissue type-specific sources and generate color-coded maps,
displaying the spatial accumulation of metabolites or tumour delimitation [1]. Dimethyl
sulfoxide (DMSO) has been reported as a potential contrast agent to evaluate GBM
response to therapy [2].
Purpose: To investigate the possibility of non-invasive therapy response assessment in
GBM through imaging of the basal and DMSO-perturbed MRSI (PE-MRSI) in
preclinical models.
Materials and Methods: C57BL/6 female mice (n=91) were inoculated with GL261
glioma cells as in [3]. MR studies were carried out at 7T, with isoflurane anaesthesia
(1.5-2%) at 37°C. The therapy consisted in three TMZ cycles [4]. Magnetic resonance
images (MRI, T2w) (TR/TEeff 4200/36ms) were acquired for tumour volume
measurement. Treated mice were studied by PE-MRSI with DMSO. A reference T2w
MRI and a 14ms TE control were acquired before DMSO injection. Parameters for
MRSI were as in [3]. Data were post-processed with 3DiCSI and MatLab to generate
time-course maps of DMSO heights and semi-supervised source-based maps [5].
Results: The sources extracted from a grid of voxels in a region of interest were able to
discriminate between GL261 tumours actively proliferating and tumours responding to
therapy, based on their metabolome pattern changes recorded by MRSI, before tumour
growth arrest was observed by MRI (Figure 1). Colour-coded nosological images
obtained throughout the course of therapy allowed convenient tracking of response
changes.
Conclusion: Source extraction and nosological imaging can serve as a valuable noninvasive tool in preclinical analysis for therapy response and monitoring.
P28
Figure 1. A) Tumour volume change in the control group (in red) of GL261 tumour bearing mice (n=16)
and one representative TMZ-treated C819 mouse (in blue). Rectangles in light green highlight the 3 TMZ
cycles. B) Representative T2w MR images (top) and nosologic color-coded maps (bottom) corresponding
to mouse C819 and MRSI grid prior to DMSO injection at chosen time points (black triangles in A)).
Blue voxels are assigned, using the semi-supervised method, to normal brain parenchyma, red voxels to
untreated/non-responding tumour and green voxels to treated responding tumour. Tumour boundaries
(T2w hiperintensity-derived) are marked with a white dotted line.
Acknowledgements: This work was funded by MINECO grant MARESCAN (SAF 2011-23870) and
Centro de Investigación Biomédica en Red - Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN,
[http://www.ciber-bbn.es/en]), an initiative of the Instituto de Salud Carlos III (Spain) co-funded
by EU FEDER. Magdalena Ciezka holds a FI-DGR grant from the Generalitat de Catalunya.
References
1.
Ortega-Martorell, S., et al., Convex non-negative matrix factorization for brain tumor
delimitation from MRSI data. PLoS One, 2012. 7(10): p. e47824.
2.
Delgado-Goni, T., et al., Dimethyl sulfoxide (DMSO) as a potential contrast agent for brain
tumors. NMR Biomed, 2013. 26(2): p. 173-84.
3.
Simoes, R.V., et al., 1H-MRSI pattern perturbation in a mouse glioma: the effects of acute
hyperglycemia and moderate hypothermia. NMR Biomed, 2010. 23(1): p. 23-33.
4.
Delgado-Goni, T., et al., DMSO-based contrast for monitoring GBM response to therapy. Magn
Reson Mater Phy MAGMA, 2011. 24(S1): p. 119-120.
5.
Ortega-Martorell S et al. A novel semi-supervised methodology for extracting tumor type-specific
MRS sources in human brain data. PLoS One. 2013 Dec 23;8(12):e83773.
P29
Kv7.1/Kv7.5 HETEROTETRAMERS WITH EMERGING PROPERTIES
ON VASCULAR SMOOTH MUSCLE PHYSIOLOGY
Anna Oliveras1, Meritxell Roura-Ferrer1,2, Laura Solé1, Alicia de la Cruz3, Angela Prieto3,
Ainhoa Etxebarria2, Joan Manils4, Daniel Morales-Cano5, Enric Condom4,
Concepció Soler4, Angel Cogolludo5, Carmen Valenzuela3, Alvaro Villarroel2,
Núria Comes1, Antonio Felipe1.
Molecular Physiology Laboratory, Departament de Bioquímica i Biologia Molecular,
Institut de Biomedicina (IBUB), Universitat de Barcelona.
2
Unidad de Biofísica, CSIC-UPV/EHU, Universidad del País Vasco.
3
Instituto de Investigaciones Biomédicas “Alberto Sols” CSIC-Universidad Autónoma de Madrid.
4
Departament de Patologia i Terapèutica Experimental, Hospital Universitari de BellvitgeUniversitat de Barcelona.
5
Departamento de Farmacología, Universidad Complutense de Madrid,
Ciber Enfermedades Respiratorias (CibeRes), Spain
1
Voltage-dependent K+ channels from Kv7 (KCNQ) family have well-established
physiological roles in cardiovascular and nervous system, although functions in
blood vessels remain unclear. Kv7.1, Kv7.4 and Kv7.5 are predominant in vascular
smooth muscle. Evidence suggests a role controlling vascular reactivity in several
smooth muscles. However, because controversial pharmacological results Kv7.1 is
under intense investigation. Therefore, establishing the entities that control smooth
muscle tone is a topic of interest. In this scenario, the ability of Kv7 channels to form
heterotetramers is of physiological relevance. Thus, the confirmation of Kv7.4/Kv7.5
heterotetramers paves the way for novel interaction that could shed light to
controversial pharmacological results. We aim whether Kv7.1 and Kv7.5 may form
heterotetrameric channels that could increase diversity on channel response in
vascular smooth muscle cells. We proved the presence of Kv7.1/Kv7.5 structures in
heterologous system by many different approaches, such as electrophysiology, coimmunoprecipitation and FRET experiments. Heteromeric channels are mainly
retained at the endoplasmatic reticulum and, unlike homomeric Kv7.1 channels,
heteromers localize out of lipid raft microdomains. These results are supported by
experiments in isolated smooth muscle myocytes. We demonstrated that Kv7.1 and
Kv7.5 are differentially expressed in aorta, cava and coronary myocytes.
Electrophysiological and miography recordings using linopiridine, chromanol 293B
and retigabine suggested that Kv7.1/Kv7.5 form heterotetramers. Coimmunoprecipitation experiments further confirmed the hypothesis. Finally, lipid raft
isolation from different tissues corroborated that predominant expression of Kv7.5
releases Kv7.1/Kv7.5 oligomers out of lipid raft structures. Our findings demonstrate
that Kv7.1 and Kv7.5 are differentially expressed in several types of blood vessels
where they contribute to control vascular reactivity. We prove that they do
heterotetramerize increasing the diversity of their physiological response. These data
may help to better understand the scenario of Kv7 channels and vascular physiology.
Supported by BFU2011-23268 and CSD2008-00005 to AF (MINECO, Spain)
P30
HOW ALLOSTERIC CONTROL OF STAPHYLOCOCCUS AUREUS
PENICILLIN-BINDING PROTEIN 2A ENABLES METHICILLINRESISTANCE AND PHYSIOLOGICAL FUNCTION
Lisandro H. Oteroa, Alzoray Rojas-Altuvea, Leticia I. Llarrullb, Cesar Carrasco-Lópeza,
Malika Kumarasirib, Elena Lastochkinb, Jennifer Fishovitzb, Matthew Dawleyb, Dusan Hesekb,
Mijoon Leeb, Jarrod W. Johnsonb, Jed F. Fisherb, Mayland Changb, Shahriar Mobasheryb
and Juan A. Hermosoa
Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física "Rocasolano",
CSIC, Serrano 119, 28006-Madrid, Spain
b
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA
a
The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical
resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus
(MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in
separate domains the transglycosylase and transpeptidase activities required for the
biosynthesis of the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the
transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of
irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the
PBP2a of MRSA is resistant to β-lactamacylation. The inability to contain MRSA
infection with β-lactam antibiotics is a continuing public health concern. We reported (1)
the identification of an allosteric binding domain (a remarkable 60 Å distant from the
DD-transpeptidase active site) discovered by crystallographic analysis of a soluble
construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational
change culminates in the opening of the active site to permit substrate entry. This same
crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid
(a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and
ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an antiMRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus
predisposing PBP2a to inactivation by a second β-lactam molecule, opens an
unprecedented realm for β-lactam antibiotic structure-based design. We will describe in
the talk the recent advances in this field.
(1) Otero et al. Proceedings of the National Academy of Sciences (2013) 110, 1680816813.
P31
INFLUENCE OF THE PHASE STATE OF E. COLI LIPID-A BILAYERS
ON THE PROPERTIES OF THE WATER/MEMBRANE INTERFACE
Krzysztof Murzyn1 and Marta Pasenkiewicz-Gierula1
Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry, Biophysics, and
Biotechnology, Jagiellonian University, Kraków, Poland
1
Lipid-A is the most conserved part of lipopolysaccharide (LPS). Both lipids form the
external layer of the outer membrane of Gram-negative bacteria. In aqueous solutions,
lipid-A alone adopts either non-lamellar or lamellar phases depending primarily on the
number of acyl chains attached to the diglucosamine backbone, temperature, hydration
level, and the presence of particular cations. Escherichia coli-specific hexa-acyl lipid-A
(ECLA) forms stable bilayers in the presence of either sodium or magnesium cations,
and its main phase transition temperature is 44°C.
Since the polymorphism of lipid A seems to play a role in many biological processes
(e.g. sepsis), the need for systematic studies of factors influencing formation of lamellar
phases by lipid-A and/or LPS is evident. Yet, due to relatively high complexity of lipidA aggregation states, their experimentally determined properties are relatively scarce.
The reported study attempts to address the problem of insufficient availability of data on
the structural properties of lipid-A bilayers by collecting and confronting the relevant
experimental results with those which are obtained from analyses of theoretical models
and simulations.
To identify and quantitatively describe the most common conformations of the lipid-A
headgroup and to characterize other biologically relevant properties of the
water/membrane interface, we performed several multi-nanosecond molecular dynamics
simulations of fully hydrated bilayers made of ECLA at 12°C and 50°C. We show that
the rate of the transition from the liquid-crystalline to the gel phase is surprisingly low
and that it can be correlated with slower mobility of ECLA molecules as compared to
phosphatidylcholines. Thanks to the assignment of carefully validated forcefield
parameters describing inter-atomic interactions of these lipids, we have determined
several structural properties of lipid-A bilayers at two different temperatures, i.e. the
surface area per lipid (SA) and the membrane width, lipid headgroup hydration pattern
as well as preferred conformations of ECLA glycosidic linkage. A thorough statistical
evaluation of differences between selected structural properties of the studied
water/membrane interfaces indicates that SA at 12°C is lower by approx. 10% than at
50°C whereas their other properties are basically unaffected.
Acknowledgements: This work is supported by the Polish National Science Center under grant no.
2011/01/B/NZ1/00081.
P32
STRUCTURE AND RNA-BINDING ACTIVITY OF THE DXV VIRUS
VP3 N-TERMINAL DOMAIN
Diego S. Ferrero1,2, Damiá Garriga1,3, Idoia Busnadiego2,4, Pablo Guerra1,
Cristina Silva1, Isabel Usón5, José F. Rodriguez2, Nuria Verdaguer1
1
Institut de Biologia Molecular de Barcelona - CSIC, Barcelona. dfecri@ibmb.csic.es
2
Centro Nacional de Biotecnología – CSIC, Madrid, España.
3
School of Biomedical Sciences, Monash University, Clayton, Australia.
4Institue of Infection, Immunity and Inflammation, University of Glasgow, Scotland.
5
Institucion Catalana de Estudis Avannçats, Barcelona.
Drosophila X virus (DXV) is a prototypic member of the Entomobirnavirus genus
within the Birnaviridae family. This family groups a number of naked icosahedral
viruses with bisegmented dsRNA genome. The Birnavirus segment A encodes a
polyprotein, which is cotranslationally cleaved to generate the capsid polypeptide pVP2
(49 kDa), the viral protease VP4 (27 kDa) and the multifunctional protein VP3 (34
kDa)[1]. Segment B encodes the RdRP VP1 (110 kDa).
The multitask protein VP3, plays an essential function in Birnavirus life cycle,
interacting with the capsid protein VP2, with the RdRP VP1 and with the dsRNA
genome. Furthermore, the role of this protein inhibiting gene silencing has been recently
demonstrated [2].
Here we report the crystal structure of the N-terminal region of DXV VP3 that contains
one of the dsRNA-binding domains (dsRBD). The recombinant purified domain,
produced in insect cells, was crystallized by the sitting drop vapour-diffusion method.
Using PEG 4K and ammonium sulphate as precipitant, needles like crystals were
obtained in space p6322, diffracting up to 2.1 Å resolution. The structure was solved by
Ab Initio methods, using program ARCIMBOLDO [3]. The VP3 dsRBD domain folds
in a bundle of three alpha helices that are arranged in a dimer, using one
crystallographic two-fold axis. PISA calculations define a monomer-monomer interface
of 1671Å2, which represents more than ~50% of the domain surface involved in dimer
formation. The basic residues of the domain appear grouped in well defined clusters.
Site directed mutagenesis analysis combined with electrophoretic mobility shift assays
and surface plasmon resonance revealed that one of the positive residues (H60) is
essential for dsRNA binding. Additionally, silencing studies performed in insect cell
cultures confirmed the importance of this dsRBD and the H60 in protein function.
Acknowledgements: This work has been sponsored by grant BIO2011-024333 to NV and AGL201124758 to JFR both from the Spanish Ministry of Economy and Competitiveness.
References
[1] A. Casañas, A Navarro, C Ferrer-Orta, D González, JF Rodriguez, N Verdaguer (2008). Structure 16:
29–37.
[2] A. Valli, I. Busnadiego, V. Maliogka, D. Ferrero, J.R. Castón, J.F. Rodriguez, J.A. García, PloS One,
2012, 7, e46768.
[3] D. Rodríguez, M. Sammito, K. Meindl, I.M. de Ilarduya, M. Potratz, GM Sheldrick, I Usón, Acta
Crystallogr, 2012, D68, 336-343.
P33
DSC BLOOD SERUM PROTEOME CHARACTERIZATION OF
MONOCLONAL GAMMAPATHY OF UNDETERMINED SIGNIFICANCE
Francisca Barceló1, Joan J. Cerdà2, Regina Alemany1, Oliver Vogler1, Antonio Gutierrez3, M.
Antonia Durán3, Antonio Novo3, Teresa Jiménez4, Teresa Ros3 and José Portugal5
Clinical and Translational Research group. University of the Balearic Islands. Palma de Mallorca, Spain.
francisca.barcelo@uib.es
2
Institute for Cross-Disciplinary Physics and Complex Systems. IFISC. UIB-CSIC, Palma de Mallorca.
Spain
3
Biología y Clínica Hematológica. Hospital Universitari Son Espases. Palma de Mallorca. Spain
4
Fundació Banc de Sang i Teixits de les Illes Balears (Gobierno Balear, Spain)
5
Instituto de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, Barcelona, Spain
1
Monoclonal gammapathy of undetermined significance (MGUS) is a premalignant
plasma cell proliferative disorder associated with a lifelong risk of progression to
multiple myeloma (MM), a malignant neoplasia. Molecular biomarkers for MGUS are
of interest for establishing markers for MGUS diagnosis and evolution. The human
plasma/serum proteome might be a suitable specimen for disease diagnosis and
therapeutic intervention. Differential scanning calorimetry (DSC) analysis of blood
plasma/serum is a useful technique to examine the proteome [1]. DSC plasma/serum
analyses have shown that thermograms from samples of healthy individuals are highly
reproducible with characteristic melting temperatures and a well-defined shape.
Thermograms obtained from several pathological diseases seem to be markedly
different from one other and this leads to a growing interest in developing DSC
technology for clinical diagnostic analysis [2]. We have examined the calorimetric
features of blood serum from patients suffering MGUS to establish the suitability of
DSC as diagnostic tool. We show that MGUS patients display thermogram profiles that
differ from those in healthy control individuals. The wide range of differences in
MGUS thermograms might be connected to the disease status and/or its progression to
MM.
Acknowledgements: This work was supported by grant BFU2010-15518 from the Spanish Ministry of
Science and Innovation and the FEDER program of the European Community.
References
[1] Nichola C. Garbett, J. J. Miller, A. B. Jenson, D. M. Miller, J. B. Chaires. Interrogation of the plasma
proteome with differential scanning calorimetry. Clin. Chem. 53, 2012-14, 2007.
[2] Nichola C. Garbett, Michael L. Merchant, C. William Helm, Alfred B. Jenson, Jon B. Klein et al.
Detection of cervical cancer biomarker patterns in blood plasma and urine by differential scanning
calorimetry and mass spectrometry. PLOS ONE 9(1), e84710, 2014.
P26
EFFECT OF WHOLE BODY HIPOTERMIA IN HUMAN LUNG
SURFACTANT BIOPHYSICS
Sonia Vázquez-Sánchez1, Mercedes Echaide1, Daniele de Luca2 and Jesús Pérez-Gil1.
Dept of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University, Madrid,
Spain. sonia.vazquez.sanchez@estumail.ucm.es
2
Division of Pediatrics and Neonatal Critical Care, FAME Dept, South Paris University Hospitals,
“A.Beclere” Medical Center, Paris, France.
1
Therapeutic whole body hypothermia (WBH) has become an evidence-based treatment
for neonates with hypoxic-ischaemic encephalopathy (HIE). Although no data exists
about the effect of WBH on humans’ lung inflammation, recent data showed better
respiratory outcomes and a trend towards lower inflammation in WBH-treated preterm
lambs. This fact suggests the potential usefulness of WBH to reduce lung injury through
the modulation of the inflammatory pathway. However, it is known that lung surfactant
behaviour highly depends on temperature. In this context, the goal of this work was to
study the effect of WBH in the behaviour of human lung surfactant to test if this
treatment could negatively affect the surfactant system.
To carry out this work, we have used bronchoalveolar lavage (BAL) obtained from
neonates with HIE who required WBH. WBH was started within the first 6h of life and
targeted at 33.5°C. Non-bronchoscopic lavage (BAL) was performed before WBH and
after 24, 48 and 72h of hypothermia.
Cell-free BAL samples were employed to study surfactant function through a
fluorescence analysis of the kinetic of adsorption into the air-liquid interface, as
previously described in Ravasio et al, 2008 [1]. Experiments were performed both at
33.5°C (WBH temperature) and at 37 °C (physiological temperature).
Surfactant adsorption seems to suffer some changes during WBH but we didn’t find
differences between assays at 33.5 and 37 ºC. Interfacial surfactant adsorption seemed
relatively impaired after the first 24 hours of hypothermia. After 48 hours, adsorption
seemed to improve, suggesting the onset of a possible adaptive compensatory
mechanism. However, samples obtained after 72 hours of hypothermia showed the most
impaired adsorption, suggesting that if there is any compensatory mechanism playing a
role, this cannot be sustained for unlimited prolonged time. In conclusion, WBH may
modulate surfactant function, although probably a time-dependent effect could be
important and deserves further studies.
Acknowledgements: This work has been sponsored by BIO2012 - 30733
References
[1] Andrea Ravasio, Antonio Cruz, Jesús Pérez-Gil, and Thomas Haller, "High-throughput evaluation of
pulmonary surfactant adsorption and surface film formation," Journal of Lipid Research 49 (11),
2479-2488 (2008).
P35
REPLICATION INITIATION PROTEINS STUDIED WITH AFM
Maria Eugenia Fuentes-Perez1, Katarzyna Wegrzyn2, Igor Konieczny2, and
Fernando Moreno-Herrero1
1
Centro Nacional de Biotecnología, CSIC, Cantoblanco, Madrid, Spain
2
University of Gdansk, Gdansk, Poland.
DNA replication is a fundamental cellular process whose mechanism is still not well
understood. Replication requires a specific DNA region, known as the origin of
replication (Ori), as well as specific proteins, called replication initiation proteins (Rep).
Both DNA and proteins form the replication initiation complex. The origin of
replication in plasmids and phage DNA contains some conserved elements. These
include specific binding sites (iterons) for Rep proteins, DnaA boxes for DnaA proteins
and an AT-rich region where DNA melting occurs. In this work, we used the Atomic
Force Microscope (AFM) to study the binding of Rep proteins to the origin of
replication in the broad-host-range plasmid RK2 [1]. The origin of replication in RK2
plasmid is called OriV. It possesses 5 iterons where the replication initiation protein
TrfA binds, four DnaA boxes for DnaA proteins and four 13-meres in the AT rich
region [2]. Using the AFM, we were able to capture the binding of TrfA to the iterons
region. Interestingly, while bound to the iterons, TrfA also interacts with a ssDNA
oligonucleotide containing the sequence of one of the strands of the AT rich region.
Moreover, the TrfA-ssDNA interaction is dependent on the sequence of the
oligonucleotide. Our AFM approach was also applied to RepE protein, a replication
initiation protein from plasmid F. Notably, we found that binding of RepE was also
favored by the equivalent ssDNA oligonucleotide of the AT-rich region of plasmid F.
These findings enable to create a general model in which firstly, Rep proteins induces
the melting of the AT-rich region and secondly, specific interaction of Rep protein with
one of the melted ssDNA occur.
Acknowledgements: This work has been supported by a Starting Grant from the European Research
Council [grant number 206117] (MEFP and FMH) and by a grant from the Spanish Ministry of Science
and Innovation [grant number FIS2011-24638] (FMH). The work in Poland was supported by the Polish
National Science Centre [grant number 24 2012/04/A/NZ1/00048] (KW and IK).
References
[1] Doran, K.S., I. Konieczny, and D.R. Helinski, Replication Origin of the Broad Host Range Plasmid
RK2. Journal of Biological Chemistry, 273(14): p. 8447-8453, 1998.
[2] Rajewska, M., K. Wegrzyn, and I. Konieczny, AT-rich region and repeated sequences–the essential
elements of replication origins of bacterial replicons. FEMS microbiology reviews, 36(2): p. 408434, 2011.
P36
EFFECTS OF MICU1 SILENCING ON MITOCHONDRIAL Ca2+ UPTAKE
Sergio de la Fuente, Jessica Matesanz-Isabel, Rosalba I Fonteriz,
Mayte Montero and Javier Alvarez
Instituto de Biología y Genética Molecular (IBGM), Departamento de Bioquímica y Biología Molecular y
Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones
Científicas (CSIC), Ramón y Cajal, 7, E-47005 Valladolid, SPAIN.
MICU1 is an important regulator of the mitochondrial Ca2+-uniporter (MCU) that has
been recently shown to act as a gatekeeper of MCU at low cytosolic [Ca2+] ([Ca2+]c).
We have studied here in detail the dynamics of MCU activity after shRNA-knockdown
of MICU1 and we find several new interesting properties. In MICU1-knockdown cells,
the rate of mitochondrial Ca2+-uptake was largely increased at low [Ca2+]c (<2µM), but
it was decreased at high [Ca2+]c (>4µM). In the 2-4µM range, a mixed behavior was
observed, where mitochondrial Ca2+-uptake started earlier in the MICU1-silenced cells
but slower than in the controls. Sensitivity of Ca2+-uptake to ruthenium red and Ru360
was similar at both high and low [Ca2+]c, indicating that the same Ca2+-pathway was
operating in both cases. The increased Ca2+-uptake rate observed at [Ca2+]c below 2µM
was transient and became inhibited during Ca2+-entry. Development of this inhibition
was slow, required 5 min for completion, and was hardly reversible. Therefore, MICU1
acts both as a MCU gatekeeper at low [Ca2+]c and as a cofactor necessary to reach the
maximum Ca2+-uptake rate at high [Ca2+]c. Moreover, in the absence of MICU1, MCU
becomes sensitive to a slow-developing inhibition that requires prolonged increases in
[Ca2+]c in the low micromolar range.
Acknowledgements: This work was supported by grants from the spanish Ministerio de Ciencia e
Innovación (BFU2011-25763) and Junta de Castilla y León (VA029A12-1). Sergio de la Fuente and
Jessica Matesanz-Isabel hold FPI (Formación de Personal Investigador) fellowships from the
Spanish Government.
References
[1] Mallilankaraman, K. et al. MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca2+
uptake that regulates cell survival. Cell 151, 630-644 (2012).
[2] de la Fuente, S. et al. Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells. Biochem.
J. 458, 33-40 (2014).
P37
TRANSMEMBRANE BUT NOT SOLUBLE HELICES FOLD
AT THE INITIAL STAGES OF PROTEIN BIOSYNTHESIS
Carlos Baeza-Delgado, Silvia Tamborero & Ismael Mingarro
Departament de Bioquímica i Biologia Molecular, Universitat de València, 46100 Burjassot, Spain
Integral membrane proteins are inserted into the ER membrane through a continuous
ribosome-translocon channel [1]. In this process, the lipid bilayer strongly constrains
polypeptide chains to adopt secondary structure conformations (mainly α-helical) to
reduce the significant free-energy penalty of embedding an exposed polar peptide
backbone into the hydrophobic core of the membrane. Currently, it is unclear to what
extent membrane-spanning regions can fold before partition from the ribosometranslocon channel into the membrane. Using glycosylation mapping as a molecular
ruler, it has been previously shown that the conformation of nascent polypeptide chains
in transit through the ribosome-translocon complex can be probed by measuring the
number of residues required to span the distance between the ribosomal P-site and the
lumenally exposed active site of the oligosaccharyl transferase enzyme [2]. With this
approach, it was possible to characterize the conformational propensities of different
model polypeptide segments inside the ribosomal exit tunnel [3]. In the present work,
we have used this technique to study the helix-forming propensities of several helices,
in order to compare the folding of transmembrane (TM) versus non-TM (soluble)
helices at the initial stages of protein biosynthesis.
Acknowledgements
This work was supported by grants BFU2009-08401 and BFU2012-39482 from the Spanish MINECO
(ERDF supported) and PROMETEO/2010/005 from the Generalitat Valenciana (to I.M.). C. B.-D. and
S.T. were recipients of predoctoral fellowship from the FPI program (MEC) and the University of
Valencia (V Segles program), respectively.
References
1. Martinez-Gil, L., Sauri, A., Marti-Renom, M. A. & Mingarro, I. (2011) Membrane protein integration
into the ER, FEBS J. 278, 3846-3858.
2. Whitley, P., Nilsson, I. M. & von Heijne, G. (1996) A nascent secretory protein may traverse the
ribosome/ER translocase complex as an extended chain, J Biol Chem. 271, 6241-6244.
3. Mingarro, I., Nilsson, I., Whitley, P. & von Heijne, G. (2000) Different conformations of nascent
polypeptides during translocation across the ER membrane, BMC Cell Biol. 1, 3.
P38
A FLUORESCENT PROBE FOR THE FtsZ ASSEMBLY SWITCH
AND THE BACTERIAL DIVISION RING
Sonia Huecas1, Laura B. Ruiz-Avila1, Marta Artola2, Lidia Araujo-Bazán1,
Erney Ramírez-Aportela1,3, Mar Martín-Fontecha2, Henar Vázquez-Villa2, Pablo Chacón3,
María L. López-Rodríguez2 and José M. Andreu1
Centro de Investigaciones Biológicas-CSIC, Madrid, Spain
sonia@cib.csic.es
2
Dpto. Química Orgánica I, Facultad de Ciencias Químicas, UCM, Madrid, Spain
3
Instituto de Química-Física Rocasolano, CSIC, Madrid, Spain
1
FtsZ is a prokaryotic structural homolog of the eukaryotic protein tubulin that assembles
forming the Z-ring, which plays a central role in bacterial cell-division, and it is a target
for new antibacterials (1). FtsZ can form single-stranded filaments with a cooperative
behavior by self-switching assembly (2), which is thought to involve the opening of a
cleft between the N-terminal and the C-terminal domains of the FtsZ monomers and can
be modified by small molecule inhibitors (3). The effective antibacterial compound
PC190723 (4) binds at this interdomain cleft of Staphylococcus aureus FtsZ and
restores MRSA sensitivity to beta-lactam antibiotics (5). PC190723 stabilizes FtsZ
filaments and condensates (6) and impairs correct Z-ring assembly (4). We have
synthesized a fluorescent analog UCM01 of PC190723 that specifically binds to FtsZ
from Bacillus subtilis and S. aureus. The binding of this probe can be conveniently
measured by fluorescence anisotropy. UCM01 does not bind to FtsZ monomers, so that
its anisotropy value increases upon FtsZ polymerization and decreases when FtsZ
disassembles. The binding of UCM01 can be displaced with increasing concentrations
of PC190723. When we add ≤50 µM UCM01 to a culture of B subtilis cells, we can
visualize the Z-ring by fluorescence microscopy without altering the normal cell
morphology and growth; when the concentration of the probe is increased, it alters cell
division as it has been previously described for benzamide derivatives (7). These results
provide the basis for further development of probes for FtsZ visualization in bacterial
cells and to identify other antibacterial ligands that could target the cleft binding site in
FtsZ.
Acknowledgements: This work has been sponsored by MINECO BFU2011-23416 and CM
S2010/BMD-2353
References
[1] Claudia Schaffner-Barbero, et al. ACS Chem Biol,7, 269-77, 2012.
[2] Sonia Huecas, et al. Biophys J, 94, 1796–1806, 2008
[3] Nathaniel L. Elsen, et al. JACS, 134, 12342-5. 2012
[4] David J. Haydon, et al. Science, 321, 1673-1675, 2008
[5] Christopher M. Tan, et al. Sci. Transl. Med., 4, 126, 126ra35, 2012
[6] José M. Andreu, et al. J Biol Chem, 285, 14239-46, 2010
[7] David W. Adams, et al. Mol Micro, 80, 68-84, 2011.
P39
BINDING OF THE BACTERIAL RepA-WH1 PRIONOID
TO MODEL LIPID VESICLES
Cristina Fernández, Mercedes Jiménez, Germán Rivas, and Rafael Giraldo
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Madrid, Spain.
Email: cfernandez@cib.csic.es
We have recently reported that engineering RepA-WH1, a bacterial DNA-toggled
protein conformational switch (dWH1  mWH1) sharing some analogies with nucleic
acid-promoted PrPc  PrPSc replication [1], constitutes a suitable synthetic model
system to study protein amyloidosis in bacteria [2, 3]. Although amyloidogenesis has
been the focus of intense research, the origin of the amyloid toxicity remains unclear.
One proposed mechanism of cytotoxicity is lipid membrane permeabilization.
In this work we have studied the aggregation of the bacterial RepA-WH1 prionoid in
the presence of cytomimetic model systems (large and giant unilamellar lipid vesicles,
LUVs, and GUVs respectively) [4]. We have observed that the interaction of RepAWH1 with membranes can catalyze aggregation. Confocal microscopy images of
protein encapsulated into GUVs show association and aggregation of the protein
preferentially to lipid vesicles containing acidic phospholipids. We have also observed
that RepA-WH1 elicits membrane disruption using a dye release assay on LUVs [5].
The extent of leakage was dependent on protein concentration.
We have been able to directly measure the process of membrane permeation and
leakage by time-elapsed imaging of dye filled GUVs upon the addition of protein. This
process is fast and over the course of the experiment most of the vesicles remain intact,
suggesting the assembly of defined pores by RepA-WH1. Knowledge of the effect of
the RepA-WH1 prionoid on membrane integrity, will provide insight into the basis for
cell death caused by amyloid proteins.
References
[1] Jerson L. Silva, Luis Mauricio T. R. Lima, Debora Foguel, Yraima Cordeiro, “Intriguing nucleicacid-binding features of mammalian prion protein”, Trends Biochem. Sci., 33, 3, 132-140, 2008.
[2] Rafael Giraldo, Susana Moreno-Díaz de la Espina, M. Elena Fernández-Tresguerres, Fátima GassetRosa, “RepA-WH1 prionoid: a synthetic amyloid proteinopathy in a minimalist host”, Prion, 5, 6064, 2011.
[3] M. Elena Fernández-Tresguerres, Susana Moreno-Díaz de la Espina, Fátima Gasset-Rosa, Rafael
Giraldo, “A DNA-promoted amyloid proteinopathy in Escherichia coli”, Mol. Microbiol. 77, 14561469, 2010.
[4] Sara M. Butterfield, Hilal A. Lashuel, “Amyloidogenic protein-membrane interactions: mechanistic
insight from model systems”, Angew Chem Int Ed. 49, 5628-5654, 2010.
[5] Bart D. van Rooijen, Mireille M.A.E. Claessens, Vinod Subramaniam, “Lipid bilayer disruption by
oligomeric α–synuclein depends on bilayer charge and accessibility of the hydrophobic core”,
Biochim. Biophys. Acta, 1788, 1271-1278, 2009.
P40
FtsZ FILAMENT DYNAMICS USING LARGE-SCALE
ATOMISTIC SIMULATIONS
Erney Ramírez-Aportela1,2, José Ramón López-Blanco1, José Manuel Andreu2, Pablo Chacón1
Department of Biological Physical Chemistry, Rocasolano Physical Chemistry Institute, CSIC,
Serrano 119. 28006 Madrid.
2
Department of Chemical and Physical Biology, Biological Research Centre, CSIC,
Ramiro de Maeztu, 9. 28040 Madrid.
1
The cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic
filaments that are essential for bacterial cell division. Our studies using unbiased
molecular simulations from representative filament crystal structures provide detailed
structural and dynamic insights into modulation of both the intrinsic curvature of the
filaments and the molecular switch coupled to the high-affinity end-wise association of
FtsZ monomers in filaments. We find different filament curvatures supported by a
nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTPFtsZ filaments bend and twist in a preferred orientation, thereby occluding the nucleotide
binding site, the differently curved GDP-FtsZ filaments exhibit a heterogeneous
distribution of open and closed interfaces between monomers. We identify a coordinated
Mg2+ ion as the key structural element in closing the nucleotide site and stabilizing GTP
filaments, whereas in GDP filaments, the loss of contact with the T7 loop of the next
monomer leads to open interfaces that are more prone to depolymerization. We
monitored, for the first time, the FtsZ monomer assembly switch, which involves
opening/closing of the cleft between the C-terminal domain and the H7 helix, and observe
the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive
conformation. These results strongly support the presence of an equilibrium between the
low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ
conformation within filaments. Finally, we find that binding of the antibiotic PC190723
suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces mimicking a non-disassembling GTP-filament.
Acknowledgements: This study was supported by grants BFU2013-44306P (P.C.), BFU 2011-23416
(J.M.A.) and CM S2010/BMD-2353 (J.M.A. and P.C.) and, by a CSIC-JAE fellowship (E.R.-A.). The
authors gratefully acknowledge the computer resources, technical expertise and assistance provided by the
Red Española de Supercomputación.
P41
DISCOVERY OF SELECTIVE LIGANDS FOR TELOMERIC RNA
G-QUADRUPLEXES (TERRA) THROUGH 19F-NMR BASED
FRAGMENT SCREENING
M. Garavís1, B. López-Méndez2, A. Somoza3, J. Oyarzabal2, C. Dalvit2,
A. Villasante4, R. Campos-Oliva2 and C. González1
Instituto de Química Física 'Rocasolano', CSIC. Serrano 119, 28006 Madrid (Spain)
2
Spectroscopy and NMR Unit & Experimental Therapeutics Programme,
Spanish National Cancer Research Center (CNIO), Melchor Fernández Almagro 3, 28029 Madrid (Spain)
3
IMDEA Nanociencia and CNB-CSIC-IMDEA Nanociencia Associated Unit
‘‘Unidad de Nanobiotecnologia’’ Cantoblanco, 28049 Madrid (Spain)
4
Centro de Biología Molecular ‘‘Severo Ochoa’’ (CSIC-UAM),
Universidad Autónoma de Madrid, Madrid (Spain)
1
Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral
target. Here we report the first successful application of 19F-NMR fragment-based
screening to identify chemically diverse compounds that bind to an RNA molecule such
as TERRA. We have built a library of 355 fluorinated fragments, and checked their
interaction with a long telomeric RNA as a target molecule. The screening resulted in
the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction
with TERRA was confirmed by 19F- and 1H-NMR as well as by CD melting
experiments. We have also explored the selectivity of the ligands for RNA Gquadruplexes, and found that some of the hits do not interact with other nucleic acids
such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel
conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition
of this particular quadruplex topology and that different ligands may recognize specific
sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting
binders promising lead compounds for fragment based drug discovery.
Acknowledgements: MICINN (CTQ2010-21567-C02-02, BFU2011-30295-C02-01, SAF2010-15440),
Comunidad Autónoma de Madrid (S2010-BMD-2457, BIPEDD2), Institutional grant from the Fundación
Ramón Areces to the Centro de Biología Molecular “Severo Ochoa”). MG was supported by the FPIfellowship BES-2009-027909.
Reference
[1] M. Garavís et al., “Discovery of selective ligands for telomeric RNA G-quadruplexes (TERRA)
through 19F-NMR based fragment screening”, ACS Chem Biol, in press, 2014.
P42
ROLE OF CDK5/CYCLIN COMPLEXES IN ISCHEMIA-INDUCED DEATH
AND SURVIVAL OF RENAL TUBULAR CELLS
Tatiana Guevara1,2, Mónica Sancho1, Enrique Pérez-Payá1,2, and Mar Orzáez1
Laboratory of Peptide and Protein Chemistry; Centro de Investigación Príncipe Felipe; Valencia, Spain
2
Instituto de Biomedicina de Valencia; IBV-CSIC; Valencia, Spain
1
Ischemia reperfusion processes induce damage in renal tubules and compromise the
viability of kidney transplants. Understanding the molecular events responsible for tubule
damage and recovery would help to develop new strategies for organ preservation. CDK5
has been traditionally considered a neuronal kinase with dual roles in cell death and
survival. Here, we demonstrate that CDK5 and their regulators p35/p25 and cyclin I are
also expressed in renal tubular cells. We show that treatment with CDK inhibitors
promotes the formation of pro-survival CDK5/cyclin I complexes and enhances cell
survival upon an ischemia reperfusion pro-apoptotic insult. These findings support the
benefit of treating with CDK inhibitors for renal preservation, assisting renal tubule
protection.
Acknowledgements: This work is dedicated to the memory of Enrique Pérez-Payá. This
work has been supported by grants from the Spanish Ministry of Science and Innovation
(MICINN-BIO2007-60066, -SAF2010-15512), and from the Generalitat Valenciana (GV)
Prometeo 2010/005 (funded in part with ERDF) to E.P.-P. T.G. was supported by a JAEpre fellowship from the Consejo Superior de Investigaciones Científicas (CSIC).
P26
IMPLEMENTATION OF A NEW ALGORITHM FOR THE DETERMINATION OF DRUGPROTEIN COMPLEX STRUCTURES COMBINING X-RAY AND NMR EXPERIMENTAL
DATA. DETERMINATION OF DACTYLOLIDE STRUCTURE BOUND TO
TUBULIN-STATHMIN COMPLEX
Javier Rodríguez Salarichs1,2, Ángeles Canales1,3, Andrea E. Prota4, J. Fernando Díaz1
Dept. Chemical and Physical Biology, Centro de Investigaciones Biológicas,
Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
2
Centro de Estudios Avanzados de Cuba, Valle Grande, La Lisa, Ciudad Habana, CP 17100, Cuba
3
Depto. Química Orgánica I. Fac. Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain
4
Biomolecular Research, Paul Scherrer Institut, Villigen PSI, Switzerland
1
X-Ray crystallography and NMR experiments are two widely used techniques in the structural
description of binding sites of protein binding drugs. The first is adequate to describe with high
resolution the structure of proteins and the drug bound when the binding constant is high enough
while the seconds is capable to describe the binding pose of protein-drug complexes with weak
association constants. Particularly, STD-NMR is useful in mapping the binding epitopes presented
by a bioactive drug to its target protein [1]–[3]. In this work we present an algorithm that combine
X-Ray and NMR techniques designed to determine the structure of protein in its bound state to a
drug when the compound binds weakly to it and it is not possible to crystallize the complex . To do
so were adapted the cost function previously used to fit STD-NMR data to particular conditions by
designing 5 new functions comparing with the previously used one [4]–[6] being the traditional
NMR R factor previously used the less accurate of all new functions designed. The best cost
function found was implemented in the algorithm employed. Finally we used the algorithm to
determine the structure of dactylolide (a potential antitumor drug) [7] bound to a tubulin-stathmin
complex [8].
Acknowledgements: We are grateful to Dr. N. R. Krishna for providing the CORCEMA-ST program and Mario Pinto
for providing the experimental STD data of kifunensine and salacinol ligands. This work was supported in part by grant
BIO2010-16351 and BQU2009-08536 from MICINN (to J.F.D. and J.J.B., respectively), BIPPED-CM from
Comunidad de Madrid (J.F.D., J.J.B., and J.M.A.), and EPSRC (I.P.).
References
[1] V. Jayalakshmi, T. Biet, T. Peters, N. R. Krishna, “Refinement of the conformation of UDP-galactose bound to
galactosyltransferase using the STD NMR intensity-restrained CORCEMA optimization”, J Am Chem Soc, 126,
8610–8611, 2004.
[2] T. Haselhorst, H. Blanchard, M. Frank, M. J. Kraschnefski, M. J. Kiefel, A. J. Szyczew, J. C. Dyason, F. Fleming,
G. Holloway, B. S. Coulson, M. von Itzstein, “STD NMR spectroscopy and molecular modeling investigation of
the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core”, Glycobiology, 17, 1030, 2007.
[3] A. Canales, J. R. Salarichs, C. Trigili, L. Nieto, C. Coderch, J. M. Andreu, I. Paterson, J. Jiménez-Barbero, J. F.
Díaz, “Insights into the interaction of discodermolide and docetaxel with dimeric tubulin. Mapping the binding
sites of microtubule-stabilizing agents using an integrated NMR and computational approach”, ACS Chem. Biol., 6,
789–799, 2011.
[4] V. Jayalakshmi, N. Rama Krishna, CORCEMA refinement of the bound ligand conformation within the protein
binding pocket in reversibly forming weak complexes using STD-NMR intensities, J. Magn. Reson. 168, 36, 2004.
[5] B. M.. Yuan, Y.a , Bleile, D.W.a , Wen, X.b , Sanders, D.A.R.c , Itoh, K.d , Liu, H.-W.d , Pinto, “Investigation of
binding of UDP-Galf and UDP-[3-F]Galf to UDP-galactopyranose mutase by STD-NMR spectroscopy, molecular
dynamics, and CORCEMA-ST calculations”, J. Am. Chem. Soc., 130, 3157–3168, 2008.
[6] J. Angulo, I. Díaz, J. J. Reina, G. Tabarani, F. Fieschi, J. Rojo, P. M. Nieto, “Saturation Transfer Difference (STD)
NMR Spectroscopy Characterization of Dual Binding Mode of a Mannose Disaccharide to DC-SIGN”,
ChemBioChem, 9, 2225–2227, 2008.
[7] J. J. Field, B. Pera, E. Calvo, A. Canales, D. Zurwerra, C. Trigili, J. Rodríguez-Salarichs, R. Matesanz, A.
Kanakkanthara, S. Wakefield, A. J. Singh, J. Jiménez-Barbero, P. Northcote, J. H. Miller, J. A. López, E. Hamel, I.
Barasoain, K. H. Altmann, J. F. Díaz, “Zampanolide, a potent new microtubule stabilizing agent, covalently reacts
with the taxane luminal site in both tubulin alpha/beta-heterodimers and microtubules”, Chem Biol, 19(6):686-98.
[8] A. E. Prota, K. Bargsten, D. Zurwerra, J. J. Field, J. F. Díaz, K.-H. Altmann, y M. O. Steinmetz, “Molecular
Mechanism of Action of Microtubule-Stabilizing Anticancer Agents”, Science, 339, 587–590, 2013.
P44
FUNCTIONAL CHARACTERIZATION OF NATIVE PULMONARY
SURFACTANT SP-B COMPLEXES SOLUBILIZED WITH DETERGENT
Marta Martínez-Calle, Bárbara Olmeda, Antonio Cruz, Begoña García-Álvarez
and Jesús Pérez-Gil
Departamento Bioquímica y Biología Molecular I, Facultad de Biología,
Universidad Complutense de Madrid. Madrid. Spain.
e-mail: martam20@ucm.es
Pulmonary surfactant is a complex mixture of lipids and specific proteins essential to
stabilize the respiratory epithelium reducing surface tension at the alveolar air-liquid
interface along the successive compression-expansion breathing cycles. Hydrophobic
surfactant proteins SP-B and SP-C are associated with lipids in surfactant membranes
and are important to maintain the proper structure and dynamics of the pulmonary
surfactant, promoting very rapid adsorption of phospholipids at the interface and respreading during expansion at inspiration.
To date, the classical method to extract and purify these proteins from the pulmonary
surfactant involves the use of organic solvents, a method which could imply disruption
of native complexes. In order to avoid this potential problem we have optimized the
purification of SP-B from native surfactant membranes through solubilization by
zwitteronic detergent CHAPS.
By performing ion-exchange chromatography with the solubilized material, cationic
protein complexes consisting mainly of SP-B were obtained. The protein showed a
similar secondary and tertiary structure than SP-B obtained by traditional solvent
purification, as revealed by circular dichroism and intrinsic fluorescence spectroscopy
assays. Functional characterization of CHAPS-solubilized SP-B was a difficult
challenge to achieve due to the surface active behaviour of the detergent traces
remaining at the sample, which could interfere with the surface tension evaluation by
captive bubble surfactometer. Improvement of detergent removal and reconstitution of
the protein into lipid membranes was therefore an important task.
Different assays were carried out to compare surface activity of SP-B solubilized by
CHAPS with classical lipid-protein complexes reconstituted from SP-B obtained in
organic solvent. The effect of several lipid-protein mixtures and different reconstitution
methods was evaluated, showing qualitatively similar surface active behaviour of both
proteins.
P45
SELF-ORGANIZATION OF THE BACTERIAL CELL-DIVISION
PROTEIN FtsZ IN CONFINED ENVIRONMENTS
Begoña Monterroso1, Sonia Mellouli2, Hanumantha Rao Vutukuri2, Esra te Brinke2,
Venkatachalam Chokkalingam2, Germán Rivas1 and Wilhelm T. S. Huck2
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain. monterroso@cib.csic.es.
Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands
1
2
We report a microfluidic approach to generate aqueous droplets of different
dimensionality in oil, stabilized as inverted micelles, to systematically probe the
polymerization of bacterial cell-division protein FtsZ into fibrous networks as a function
of the concentrations of crowding agent and FtsZ. Microdroplets in microfluidics are
completely monodisperse and easily manipulated inside microfluidic channels, and
represent different topological compartments amenable for the study of different
biological processes of interest [1]. The wide knowledge derived from the previous
biophysical characterization of FtsZ [2] has been decisive to achieve its successful
encapsulation into lipid-coated water-in-oil droplets that allowed studying the effect of
crowding and confinement on the spatial distribution of FtsZ bundles [3]. The surface of
the droplets was composed by the ternary mixture of E. coli lipids, while intracellular
crowding was induced with Ficoll 70. It was determined that the distribution of the
polymer network inside the droplets was a result of the geometry of the container,
inducing in turn different confinement effects, and that surface and/or crowder
composition greatly affected the processes occurring inside the droplets. The turnover of
subunits, as determined by fluorescence recovery after photobleaching (FRAP), was
similar to that previously determined in vivo, indicating the experimental conditions
within the droplet resemble the environment in the cytoplasm of the cell. Current efforts
are directed towards the reconstruction inside droplets of FtsZ with its companion
proteins and site-selection systems through which the bacteria controls the division
process, reproducing the crowded intracellular media and modulating also other factors
potentially affecting the system reactivity.
Acknowledgements: B.M and G.R. were supported by the Spanish government (BIO2011-28941-C0303), the European Commission (HEALTHF3-2009-223432), the Human Frontiers Science Program
(RGP0050/2010-C102) and the Comunidad de Madrid (S-BIO-0260/2006). W.T.S.H. acknowledges
funding from the European Research Council (ERC Advanced grant Intercom 246812) and the
Netherlands Organization for Scientific Research (NWO, VICI award).
References
[1] A.B. Theberge, F. Courtois, Y. Schaerli, M. Fischlechner, C. Abell, F. Hollfelder, W.T. Huck,
“Microdroplets in Microfluidics: An Evolving Platform for Discoveries in Chemistry and Biology”,
Angew. Chem., Int. Ed., 49, 5846–5868. 2010.
[2] B. Monterroso, C. Alfonso, S. Zorrilla, G. Rivas, “Combined analytical ultracentrifugation, light
scattering and fluorescence spectroscopy studies on the functional associations of the bacterial
division FtsZ protein” Methods 59, 349-362. 2013.
[3] S. Mellouli, B. Monterroso, R. Vutukuri, E. Brinke, V. Chokkalingam, G. Rivas, W.T.S. Huck ,
“Self-organization of the bacterial cell-division protein FtsZ in confined environments” Soft Matter
9, 10493-10500. 2013.
P26
EFFECT OF SALT, NUCLEOTIDES AND MACROMOLECULAR CROWDING ON
THE SELF-ASSOCIATION OF ClpB PROTEIN: AN ANALYTICAL
ULTRACENTRIFUGATION AND LIGHT SCATTERING STUDY.
Carlos Alfonso1, Urko del Castillo2, Ianire Martín2, Sergio P. Acebrón2, Garbiñe Celaya2, Ariadna
Martos3, Fernando Moro2, Germán Rivas1, and ArturoMuga2
Centro de Investigaciones Biológicas (CSIC), E-28040 Madrid (Spain).
carlosa@cib csic.es
2
Biophysics Unit (CSIC) and Department of Biochemistry & Molecular Biology,
Basque Country University, E- 48080 Bilbao (Spain).
3
Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried (Germany)
1
ClpB is an oligomeric molecular chaperone that, together with the DnaK system, has the ability
to disaggregate stress-denatured proteins [1]. We studied how the self-association of ClpB
(hexameric in its active form) is regulated by salt and physiological nucleotides and also by a
deletion of its M domain. We combined biochemical assays with analytical ultracentrifugation
and composition-gradient static light scattering (CG-LS) [2]. Wild type ClpB is mainly
hexameric at low salt concentration (50 mM NaCl) in all the concentration range that was
analysed (1-40 μM) and monomeric in high salt buffer (500 mM KCl) and low protein
concentration. The dissociation of ClpB hexamers begins to be observable above 150 mM KCl
and it is completed at ~ 400 mM KCl. The M mutant ClpB begins to dissociate at 10 μM, which
is a clear demonstration that the mutation affects the stability of hexamer [3].
In a second study we have compared the association equilibrium, biochemical properties,
stability and chaperone activity of wild type ClpB in the absence and presence of an inert
macromolecular crowding agent [4]. Our data show that crowding i) increases 3-4 orders of
magnitude the association constant of the functional hexamer, ii) shifts the conformational
equilibrium of the protein monomer toward a compact state; iii) stimulates its ATPase activity;
and iv) favors association of the chaperone with substrate proteins and with aggregatebound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaKClpB network, highlighting the importance of volume exclusion in complex processes in which
several proteins have to work in a sequential manner.
Acknowledgements: This work was supported by the Ministerio de Educación y Ciencia (Grants BFU200764452 and BFU2010-15443 to A. Muga, and BIO2008-04478-C03-03 and BIO2011-28941-C03 to G. Rivas),
the Universidad del País Vasco and Gobierno Vasco (Grants IT-358-07 and IT709-13) and Diputación Foral de
Bizkaia (Grant DIPE08/19).
References
[1] Weibezahn J.,Schlieker C., Tessarz P., Mogk A, Bukau B. “Novel insights into the mechanism of chaperoneassisted protein disaggregation”. J. Biol. Chem. 386, 739-44 (2005).
[2] Attri A.K., Minton A.P. “New methods for measuring macromolecular interactions in solution via static light
scattering: basic methodology and application to nonassociating and self-associating proteins”. Anal.
Biochem. 337, 103-10 (2005).
[3] Del Castillo U., Alfonso C., Acebrón S.P., Martos A, Moro F, Rivas G, Muga A. “A quantitative analysis of
the effect of nucleotides and theM domain on the association equilibrium of ClpB”. Biochemistry 50, 1991–
2003 (2011).
[4] Rivas G., Minton A.P. “Beyond the second virial coefﬁcient: Sedimentation equilibrium in highly non-ideal
solutions”. Methods 54, 167–174 (2011).
P47
CHARACTERIZATION OF A NEW CHEMOTYPE OF
MICROTUBULE MODULATOR OF THE VINCA DOMAIN
Gonzalo Sáez-Calvo1, Ashwani Sharma2, Mariano Redondo-Horcajo1, Ruth Matesanz1, Ángeles
Canales1, Katja Bargsten2, Andrea E. Prota2, Clemens Lamberth3, Sebastian Wendeborn3, Isabel
Barasoain1, Michel O. Steinmetz2 and José Fernando Díaz1
Centro de Investigaciones Biológicas (CIB-CSIC), Physical and Chemical Biology Department.
Madrid, Spain. gonsaez@cib.csic.es
2
Paul Scherrer Institut, Laboratory of Biomolecular Research. Villigen, Switzerland
3
Syngenta Crop. Protection AG, Research Department, Schaffhauserstr. 101, CH-4332 Stein, Switzerland
1
Cancer is one of the leading causes of death worldwide accounting for eight million
deaths each year [1]. One of major targets in cancer chemotherapy research is tubulin
which plays a key role in processes involved in cancer grow and metastasis [2].
Vinblastine and Paclitaxel (tubulin binders), were widely used anti-tumour agents.
Nevertheless, its use [3] has shown that tumoural cells are able to generate resistance
(during treatment) against these drugs through overexpression of cellular pumps, like PGlycoprotein, that expel drugs out of the cell [4]. This is why, nowadays, efforts are
focused on design of new antitumor agents able to overcome this resistance either though
high affinity for the target [5] or though low affinity for PGP.
CSCD062122 is a new compound which targets microtubule network. Through the use
of various biophysical, biochemical and biological techniques, the binding of this
compound and a series of derivatives have been characterized.
Cell biology analysis indicates that these compounds avoid P-glycoprotein resistance
becoming then a promising chemotype to be explored. Exposition of tumoural cells to the
compound induces micrtotubule bundles and multipolar mitosis, as expected for a tubulin
binder.
CSCD062122 is not able to bind in vitro neither dimeric tubulin nor microtubules, but it
does it to tubulin tetramers in solution with a 0.5:1 stoichiometry suggesting that the
compound is an interfacial ligand.
At low ligand concentrations, CSCD062122 induces the formation of tubulin-oligomers,
instead of microtubules as expected for an interfacial ligand. As revealed by TEM and
SAXS experiments, these oligomers are ring-like structures of roughly thirty nanometers
in diameter.
We have also performed structural studies using NMR which indicated the binding
epitope and using X-ray crystallography, the Tubulin2-RB3-ligand crystal confirmed
superposition with the vinblastine site [6].
From the comparison of the structure obtained with this of the Tubulin2-RB3-vinblastine
[6] it is possible to conclude that the CSCD062122 mechanism of action involves binding
into the interdimer interface, however the structural effect of the compound binding
involves a modification of the angle of contact between the tubulin dimers inducing a
P47
straightened structure, which could explain the existing differences in the tubulin
aggregates observed.
Acknowledgements: This work has been sponsored by a FPI Grant from the Spanish Ministry of
Economy and Competitiveness (BES-2011-043408) depending on the National Plan of Research BIO2010-16351.
References
[1] World Health Organization webpage (WHO), www.who.int/en.
[2] Phan L. M., Yeung S. C. and Lee M. H. “Cancer metabolic reprogramming: Importance, main
features and potentials for precise targeted anti-cancer therapies”, Cancer Biol. Med., 11, 1, 1 – 19,
2014.
[3] Ginsberg J., “The discovery of Camptothecin and Taxol” (National Historic Chemical Landmarks
program of the American Chemical Society, 2003).
[4] Yamagishi T., Sahni S., Sharp D. M., Arvind A., Jansson P. J. and Richardson D. R. “P-glycoprotein
mediates drug resistance via novel mechanism involving lysosomal sequestration”, J. Biol. Chem.,
288, 44, 31761 – 71, 2013.
[5] Martínez-Díez M., Guillén-Navarro M. J., Pera B., Bouchet B. P., Martínez-Leal J. F., Barasoain I.,
Cuevas C., Andreu J. M., García-Fernández l. F., Díaz J. F., Avilés P and Galmarini C. M.
“PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein
over-expressing tumors.”, Biochem. Pharmacol., 88, 3, 291 – 302, 2014.
[6] Gigant B., Wang C., Ravelli R. B., Roussi F., Steinmetz M. O., Curmi P. A., Sobel A. and Knossow
M. “Structural basis for the regultation of tubulin by vinblastine”, Nature, 435, 7041, 519 – 22, 2005.
P48
THE TRANSMEMBRANE DOMAIN INTERACTION NETWORK
OF Bcl-2 FAMILY MEMBERS
Vicente Andreu-Fernández1, Ainhoa Genovés1, Ismael Mingarro2 , Mar Orzáez1 and
Enrique Pérez Payá
Centro de Investigación Príncipe Felipe, Valencia, España;
vandreu@cipf.es
2
Universidad de Valencia, Valencia, España.
1
Intrinsic apoptosis in mammals is regulated by protein-protein interactions among the
B-cell lymphoma-2 (Bcl-2) family1. The relevance of Bcl’s BH3 cytosolic domain in
defining the protein-protein interactions between different pro- and anti- apoptotic
members has been extensively analyzed2. However, the function of the transmembrane
domain (TMD) in such interactions has not been yet defined. It is becoming apparent
that the TMDs are more than mere insertion domains and may play a key role in the
function of the Bcl-2 proteins3. We have analyzed the contribution of TMDs to the
regulation of protein interactions among the different pro- and anti-apoptotic Bcl-2
members and their relevance to the control of the apoptotic cellular fate. To accomplish
these objectives we have performed an overall study of the TMD protein-protein
interactions among the different Bcl-2 proteins4,5. The results of this study have
prompted us to define the Bcl-2 TMD interaction map.
Acknowledgements: This work has been sponsored by grants from the Spanish Ministry of Science and
Innovation (MICINN-BIO2007-60066, -SAF2010 15512, -SAF2008-00048 and CSD2008-00005C), and
by Generalitat Valenciana (GV) Prometeo 2010/005.
References
1. Llambi F, Moldoveanu T, Tait SW, Bouchier-Hayes L, Temirov J, McCormick LL, Dillon CP,
Green DR. A unified model of mammalian BCL-2 protein family interactions at the
mitochondria. Mol Cell; 44(4):517-31. (2011).
2. Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR. The BCL-2 family reunion. Mol
Cell; 37(3):299-310. (2010).
3. Lindsay J, Esposti MD, Gilmore AP. Bcl-2 proteins and mitochondria Specicity in membrane
targeting for death. Biochimica et Biophysica Acta 1813, 532–539 (2011).
4. Yin H, Slusky JS, Berger BW, Walters RS, Vilaire G, Litvinov RI, Lear JD, Caputo GA, Bennett
JS, DeGrado WF. Computational Design of Peptides That Target Transmembrane Helices.
Science; 315(5820):1817-22 (2007).
5. Zhu H, Metcalf DG, Streu CN, Billings PC, Degrado WF, Bennett JS. Specificity for
homooligomer versus heterooligomer formation in integrin transmembrane helices. J Mol Biol.
401(5):882-91 (2010).
P49
STRUCTURAL BASIS FOR THE MODULATION OF TUBULIN
BY ANTITUMORAL DRUGS
J. Fernando Díaz1, Andrea Prota2 and Michel O. Steinmetz2
Centro de Investigaciones Biológicas (CIB-CSIC), Physical and Chemical Biology, Madrid, Spain
fer@cib.csic.es
2
Paul Scherrer Institut, Laboratory of Biomolecular Research, Villigen, Switzerland
1
Tubulin modulators work by altering the activation state of tubulin, either by deactivating
the active GTP-bound molecule or activating the otherwise inactive GDP-bound
molecule. We have successfully crystallized the ternary complexes formed between the
Tubulin2-RB3 and several microtubule modulating drugs in three different regulatory
sites, this of the paclitaxel site (1), the lauli/peloruside site (2) and a newly described site
to which maytansine, rhizoxin or the Phase I drug PM060184 (3) bind finding the
structural determinants modified in tubulin by these drugs and responsible for the
activation (paclitaxel, laulimalide/peloruside sites) or deactivation (maytansine site).
Ligands of the paclitaxel site bind to a hydrophobic cavity in inner lumen of the
microtubules (4). Despite the importance of paclitaxel for medical applications and basic
research, their molecular mechanisms of action on tubulin and microtubules remain
elusive. We determined high-resolution crystal structures of -tubulin in complex with
two unrelated MSAs, zampanolide and epothilone A (5). Both compounds were bound to
the taxane pocket of -tubulin and used their respective side chains to induce structuring
of the M-loop into a short helix. Because the M-loop establishes lateral tubulin contacts
in microtubules, these findings explain how taxane-site MSAs promote microtubule
assembly and stability.
Secondly we have found using X-ray crystallography that laulimalide and peloruside A
bind to a unique non-taxane site on -tubulin and use their respective macrolide core
structures to interact with a second tubulin dimer across protofilaments (6). At the same
time, they allosterically stabilize the taxane-site M-loop that establishes lateral tubulin
contacts in microtubules. Structures of ternary complexes of tubulin with
laulimalide/peloruside A and epothilone A are also solved.
Finally we have found that Maytansine rhizoxin or the Phase I drug PM060184 bind to
an unprecedented site on ‐tubulin and blocks the formation of longitudinal tubulin
interactions in microtubules.
Our results explain the molecular mechanism of action of clinically relevant microtubule
modulating agents. They further provide a structural basis for the rational design of highly
potent microtubule modulating agents, opening a possible route for the development of
next‐generation drugs for the treatment of cancer.
Acknowledgements: This work was supported by grants from the Ministerio de Economía y
Competitividad (BIO2010‐16351 to JFD) and the Comunidad Autónoma de Madrid (S2010/BMD‐
2457 to JFD) and by a grant from the Swiss National Science Foundation (310030B_138659; to MOS)
P49
References:
1. Parness, J., and Horwitz, S. B. (1981) Taxol binds to polymerized tubulin in vitro, J Cell Biol 91, 479487.
2. Pryor, D. E., O'Brate, A., Bilcer, G., Díaz, J. F., Wang, Y., Kabaki, M., Jung, M. K., Andreu, J. M.,
Ghosh, A. K., Giannakakou, P., and Hamel, E. (2002) The microtubule stabilizing agent laulimalide
does not bind in the taxoid site, kills cells resistant to paclitaxel and epothilones, and may not require
its epoxide moiety for activity, Biochemistry 41, 9109-9115.
3. Pera, B., Barasoain, I., Pantazopoulou, A., Canales, A., Matesanz, R., Rodriguez-Salarichs, J., GarciaFernandez, L. F., Moneo, V., Jimenez-Barbero, J., Galmarini, C. M., Cuevas, C., Penalva, M. A., Diaz,
J. F., and Andreu, J. M. (2013) New Interfacial Microtubule Inhibitors of Marine Origin,
PM050489/PM060184, with Potent Antitumor Activity and a Distinct Mechanism, ACS Chemical
Biology 8, 2084-2094.
4. Nogales, E., Whittaker, M., Milligan, R. A., and Downing, K. H. (1999) High-resolution model of the
microtubule, Cell 96, 79-88.
5. Prota, A. E., Bargsten, K., Zurwerra, D., Field, J. J., Diaz, J. F., Altmann, K. H., and Steinmetz, M. O.
(2013) Molecular mechanism of action of microtubule-stabilizing anticancer agents, Science 339, 587590.
6. Prota, A. E., Bargsten, K., Northcote, P., M., M., Altmann, K. H., Miller, J. H., Díaz, J. F., and
Steinmetz, M. (2014) Structural basis of microtubule stabilization by laulimalide/peloruside and their
synergy with taxane site anticancer drugs, Angew Chem Int Ed Engl 53(6):1621-5.
7.- Prota, A.E, Bargsten, K., Diaz, J.F., May Marsh, M. Cuevas, C., Liniger, M., Neuhaus, C., Andreu,
J.M. Altmann K.-H. and Steinmetz, M. O. A new tubulin‐binding site and pharmacophore for
microtubule‐destabilizing anticancer drugs. Submitted
P50
BIOPHYSICAL EVALUATION OF PULMONARY SURFACTANT
AS A DRUG DELIVERY SYSTEM
Alberto Hidalgo, Antonio Cruz and Jesús Pérez-Gil
Department of Biochemistry, Faculty of Biology, Complutense University, Madrid, SPAIN.
albertohidalgo@ucm.es
The discovery of new therapeutic agents includes the development of innovative drug
delivery strategies. In this sense, the respiratory surface is in principle a perfect site for
drug entry, not only intended for local but also for systemic treatments, due to unique
features including its large surface area (100m2 approx.), the high permeability of its
membranes and the large vascularity, as well as the thin alveolar epithelium by which
the respiratory surface is covered. Besides, a low enzymatic activity ensures very low
clearance of drugs and nanoparticles [1]. Consequently, absorption and bioavailability
of different types of molecules are considerably higher than delivered through
conventional ways (oral or topical), especially those that are poorly water-soluble.
To consider lungs as a target for drug delivery, it is essential to take into account that
the respiratory surface of the mammalian lung is covered by a thin aqueous layer, and
on top of it, by a lipid-protein surface active material, the pulmonary surfactant (PS).
The main function of this surfactant is to prevent pulmonary collapse during breathing,
reducing surface tension at the air-liquid interface below 5mN/m [2]. Its particular
composition and structure provides PS with unique biophysical properties, which
facilitate its rapid adsorption into the air-liquid interface and a very efficient spreading
along it. Accordingly, PS could act as an efficient shuttle for drug delivery. Both the
biophysical properties of PS and the advantages of the respiratory system could be used
for delivering and transporting efficiently different types of drugs and nanoparticles
along the whole pulmonary surface.
In this work we have analysed the interaction of different molecules with PS and its
carrier capacity. We have firstly focused on determining how different molecules can be
incorporated into PS complexes and how this depends on PS composition, including the
presence or absence of cholesterol. Then, we have evaluated the impact of incorporated
molecules on surfactant function and mechanical properties, as assessed in a captive
bubble surfactometer. Finally, we have designed and optimized a novel setup to
evaluate the spreading capabilities of surfactant as well as the carried molecules along
the air-water interface.
References:
[1] Weber, S., Zimmer, A., Pardeike, J. “Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid
Carriers (NLC) for pulmonary application”. Eur J Pharm Biopharm. 2014;86(1):7-22.
[2] López-Rodríguez, E., Pérez-Gil, J. “Structure-function relationships in pulmonary surfactant
membranes: From biophysics to therapy”. Biochim Biophys Acta. 2014;1838(6):1568-1585.
P51
NANOMECHANICS OF CELL-SURFACE PROTEIN CD4
Alvaro Alonso-Caballero1 and Raul Perez-Jimenez1
CIC nanoGUNE Consolider, Donostia-San Sebastian, Spain
a.alonso@nanogune.eu
1
In this research project we study how forces affect the cell-surface protein CD4, a
transmembrane protein which extracellular part is composed by four immunoglobulin
domains. Besides its role in adaptive immunity, CD4 is involved in the attachment of
the human immunodeficiency virus (HIV-1) to the surface of lymphocyte T cells1. Most
of the research done about viral infection has been centered in the biochemical and
cellular processes that take place during infection2. Here, we use single-molecule atomic
force spectroscopy (AFS) to study the mechanical response of the first two domains of
CD4, D1 and D2, when a force is applied. We use force-extension (FX) atomic force
microscopy for applying constant pulling speed to the protein, monitoring the unfolding
of CD4 domains. With FX we were able to reach very low speeds (10 nm/s) that could
likely resemble the ones that the protein experience in vivo. Our results indicate that D2
domain unfolds before D1, but the unfolding force is bigger in D2 (74±24 pN) than D1
(57±21 pN) indicating a hierarchical behavior in the mechanical properties of
CD4D1D2. In addition, we have carried out force-clamp (FC) experiments, in which we
pull the protein at a constant force. We monitored the mechanical stability of the protein
at forces from 20 to 100 pN showing that mechanical extension occurs even at very low
forces. We hypothesized that the interaction of the virus particle with CD4 domains
triggers mechanical conformational changes that might be important for the infection
process. This study represents the first investigation of the mechanics of viral receptors
and brings a new scenario to be considered for a better understanding of the molecular
aspects of viral infections.
References:
[1] Craig B. Wilen, John C. Tilton and Robert W. Doms “HIV: Cell Binding and Entry”, Cold Spring
Harb Perspect Med 2012; 2:a006866
[2] Craig B. Wilen, John C. Tilton and Robert W. Doms “Viral Molecular Machines” (Springer US.,
2012).
P52
MECHANICS OF CONSTRICTION DURING CELL DIVISION:
A VARIATIONAL APPROACH
Victor G. Almendro-Vedia1, Francisco Monroy2 and Francisco J. Cao3
Departamento de Química Física I, Universidad Complutense de Madrid, Madrid, Spain.
Departamento de Química Física I, Universidad Complutense de Madrid, Madrid, Spain.
3
Departamento de Física Atómica, Molecular y Nuclear,
Universidad Complutense de Madrid, Madrid, Spain
francao@fis.ucm.es
1
2
During symmetric division cells undergo large constriction deformations at a stable
midcell site. Using a variational approach, we investigate the mechanical route for
symmetric constriction by computing the bending energy of deformed vesicles with
rotational symmetry. Forces required for constriction are explicitly computed at
constant area and constant volume, and their values are found to be determined by cell
size and bending modulus. For cell-sized vesicles, considering typical bending modulus
of κ ~ 10kBT, we calculate constriction forces in the range 0.1-1pN. The instability of
symmetrical constriction is shown and quantified with a characteristic coefficient of the
order of -50kBT, thus evidencing that cells need a robust mechanism to stabilize
constriction at midcell.
Acknowledgements: This work was supported by Ministerio de Ciencia e Innovación and Ministerio
de Economía y Competitividad (Spain) under grants FIS 2010-17440 to FJC and FIS2009-14650C02-01, FIS2012-35723, Consolider Ingenio en Nanociencia Molecular CSD2007-0010, and
S2009MAT-1507 from Comunidad Autonoma de Madrid (Spain) to FM. VGAV acknowledges
support from Ministerio de Educación Cultura y Deporte (Spain) through Becas de Colaboración
program. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
References
[1] Almendro-Vedia VG, Monroy F, Cao FJ, “Mechanics of Constriction during Cell Division: A
Variational Approach”, PLoS ONE 8, e69750, 2013.
[2] Weiss P, “From cell to molecule. The Molecular Control of Cellular Activity”, (McGraw-Hill,
1962).
[3] Boal D, “Mechanics of the Cell”, (Cambridge University Press, 2002).
[4] Seifert U, Berndl K, Lipowsky R, “Shape transformations of vesicles: phase diagram for
spontaneous curvature and bilayer coupling model”, Phys Rev A, 44: 1192–1202, 1991.
[5] Chen IA, “Cell division: breaking up is easy to do”, Curr Biol, 19: R327–R328, 2009.
P53
MECHANICS OF CONSTRICTION DURING CELL DIVISION:
APPROXIMATIVE ANALYTICAL RESULTS FOR SYMMETRIC
CONSTRICTION
Victor G. Almendro-Vedia1, Francisco Monroy2 and Francisco J. Cao3
Departamento de Química Física I, Universidad Complutense s/n, Madrid, Spain
vgavedia@fis.ucm.es
2
Departamento de Química Física I, Universidad Complutense s/n, Madrid, Spain
3
Departamento de Física Atómica y Molecular, Universidad Complutense s/n, Madrid, Spain
1
In this work approximate analytical expressions are provided for the main magnitudes
of a symmetrically constricted vesicle. These magnitudes include constriction energy,
length of the constriction zone, volume and area of the vesicle, and the stability
coefficient for symmetric constriction. The analytical expressions are derived
combining a perturbative expansion in the lagrangian for small deformations with a
cosine ansatz in the constriction region.
Acknowledgements: This work was supported by Ministerio de Ciencia e Innovación and Ministerio
de Economía y Competitividad (Spain) under grants FIS 2010-17440 to FJC and FIS2009-14650C02-01, FIS2012-35723, Consolider Ingenio en Nanociencia Molecular CSD2007-0010, and
S2009MAT-1507 from Comunidad Autonoma de Madrid (Spain) to FM. The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
[1] Seifert U, Berndl K, Lipowsky R, “Shape transformations of vesicles: phase diagram for
spontaneous curvature and bilayer coupling model”, Phys Rev A, 44: 1192–1202, 1991.
[2] Seifert U, Lipowsky R, “Morphology of Vesicles. Handbook of Biological Physics vol. I”, (Elsevier
Science, 1995).
[3] Surovtsev IV, Zhang Z, Lindahl PA, Morgan JJ, “Mathematical modeling of a minimal protocell
with coordinated growth and division”, J Theor Biol, 2009.
[4] Boal D, “Mechanics of the Cell”, (Cambridge University Press, 2002).
P54
A POTENTIAL ROLE OF I-MOTIFS IN CENTROMERIC CHROMATIN
ORGANIZATION: THE CENTROMERIC ALPHA-SATELLITE DNA IS ABLE
TO FORM I-MOTIF STRUCTURES
Miguel Garavís1,2, Nuria Escaja4, Diana Carreras1, Valérie Gabelica3,
Alfredo Villasante2 and Carlos González1
Instituto de Química Física Rocasolano, CSIC. Serrano 119, 28006 Madrid (Spain)
2
Centro de Biología Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Madrid (Spain)
3
Univ. Bordeaux, ARNA Laboratory, IECB, 2 rue Robert Escarpit F-33600 Pessac (France)
4
Dept. Química Orgànica. Universitat de Barcelona. C/. Martí i Franquès 1-11, 08028 Barcelona. Spain
1
The centromere is the chromosomal region where the kinetochore forms and attaches to
spindle microtubules to ensure proper chromosome segregation. The centromeres of
eukaryotic chromosomes are formed by tandemly repeated “satellite” DNA sequences
that evolve rapidly by recombination processes without losing their function (centromere
“paradox”). Since human centromeres have been extensively investigated, their alphasatellite DNA is an excellent model to understand the role of satellite DNAs in centromere
structure and function.
In humans there are two types of alpha-satellite monomers, named type A and type B.
The differences between A and B types are concentrated in a 17 bp region, called the
A/B-box. The phasing of centromeric nucleosomes on alpha-satellite DNA places the Aand the B-box at the entrance and exit of the nucleosome. In vitro, the C-rich strand of
the B-box adopts a dimeric i-motif structure stabilized by five intercalated hemiprotonated C:C+ base pairs, observable even at nearly neutral pH.1
In this communication we show that the A-box of the alpha-satellite DNA is also able to
form dimeric i-motif structures in vitro, suggesting the possibility that this non-canonical
DNA structure may have a role in the structural organization of the centromere. We
propose that centromeric satellites may have been selected for their ability to form i-motif
structures, and not on the basis of their primary sequence, as is generally believed. This
stable i-motif topology may confer an enhanced resistance against the pulling forces felt
by the centromeric chromatin during chromosome segregation.
Acknowledgements: MICINN (CTQ2010-21567, BFU2011-30295-C02-01). MG was supported by the
FPI-fellowship BES-2009-027909
References:
[1] J. Gallego, S.H. Chou, and B.R. Reid“Centromeric pyrimidine strands fold into an intercalated motif
by forming a double hairpin with a novel T:G:G:T tetrad: solution structure of the d(TCCCGTTTCCA)
dimer”, J Mol Biol, 273, 840-856, 1997.
P55
NANOMECHANICAL EVOLUTION OF THE
GIANT ELASTIC PROTEIN TITIN
Aitor Manteca1 and Raul Perez-Jimenez1, 2
1
2
CIC nanoGUNE Consolider. Tolosa Hiribidea 76, 20018 Donostia (Spain)
IKERBASQUE. Basque Foundation for Science. Alameda Urquijo, 36-5, 48011 Bilbao (Spain)
Titin is a gigantic pseudoelastic muscular protein responsible for the elasticity, integrity
and signal transduction of the muscle filaments. This protein is present in all animals
differentiating them from other organisms. One of the major components of titin is the
array of immunoglobulin domains that confer spring-like behavior. Nevertheless, it is
unknown how titin domains have achieved their remarkable mechanical properties.
Molecular evolution and nanomechanical analysis of this protein has been carried out by
means of phylogenetic studies and single-molecule atomic force spectroscopy
measurements. Combining biostatical tools and molecular biology procedures we were
able to predict and bring back to life titin structures up to 300 million year old [1]. Force
extension atomic force spectroscopy results show two differentiated populations in I65 I72, an eight immunoglobulin-like domain construct of titin located within the I-Band.
Concretely, human titin I65 - I72 contour length measurements exhibit one population
at 31 ± 2 nm (93% of events) with and average force of 231 ± 71 pN and another
population at 8.3 ± 1.6 nm (7% of events, average force 183 ± 31 pN). On the other
hand, LTCA (Last Tetrapod Common Ancestor, 325 million year old) I65 - I72 show
similar populations: one at 31.5 ± 1.7 nm (80% of events, average force 216 ± 42 pN)
and another one at 8.4 ± 1.7 nm (20% of events, average force 157 ± 35 pN). The main
differences between the constructs are the percentage of events in the populations and
the average force of the domains, which is around 15% lower in LTCA. We have found
that ancestral titin domains show lower mechanical stability than that of human titin
domains and some of them contain unfolding lengths consistent with the presence of
disulfide bonds. This is in agreement with the fact that the ancestral sequence contains a
higher number of cysteine residues than that of human titin. These findings suggest that
disulfide bonds were mechanical regulators in Titin that have been substituted by
hydrogen bonds over the course of evolution producing more extensible and flexible
proteins. We hypothesize that this substitution derived in physiological changes that
allowed the muscular development and morphological diversity of modern animals.
Acknowledgements: This work has been sponsored by CIC nanoGUNE Consolider
References
[1]
Raul Perez-Jimenez, Alvaro Inglés-Prieto, Zi-Ming Zhao, Inmaculada Sanchez-Romero, Jorge
Alegre-Cebollada, Pallav Kosuri, Sergi Garcia-Manyes, T Joseph Kappock, Masaru Tanokura,
Arne Holmgren, Jose M Sanchez-Ruiz, Eric A Gaucher & Julio M Fernandez et al. “Singlemolecule paleoenzymology probes the chemistry of resurrected enzymes”, Nat Struct Mol Biol,
18, 592–596, 2011.
P56
FUNCTIONAL UP-MODULATION OF PLASMA MEMBRANE Ca2+-ATPASE
BY METHYLENE BLUE
Ana M. Mata, María Berrocal and Isaac Corbacho
Depto. Bioquímica y Biología Molecular y Genética, Facultad Ciencias, Universidad de Extremadura,
Avda de Elvas s/n, 06006 Badajoz, Spain.
The activity of hPMCA4 isoform overexpressed in COS cells is enhanced by methylene
blue (MB), a phenotiazine cationic dye that has been evaluated in Phase 2 and Phase 3
Alzheimer's disease (AD) trials. We have seen that PMCA activity is partially inhibited
by the neurotoxic amyloid-β peptide (Aβ). In this work we show that this inhibition is
completely blocked by MB in the human intact isoform and also in pig brain purified
preparation and human brain membranes not affected by AD. In order to determine
protein-dye binding sites we prepared two mutants (hPMCA4b-L1086* and hPMCA4bR1052*) lacking the calmodulin binding domain and the whole cytosolic C-tail of PMCA,
respectively. Activity assays showed that both truncated forms were not affected by Aβ
while only the hPMCA4b-L1086* mutant was activated by MB. Therefore, the MB dye
seems to block the inhibitor effect of Aβ on PMCA by its binding to a site close to the
last transmembrane domain. The MB effect on PMCA may be of potential interest by its
ability to induce a conformational change on the protein that enhance its key function in
cytosolic Ca2+ pumping, thus removing the excess of calcium accumulated in the cytosol
in neurodegenerative pathologies as AD.
Acknowledgements: This work has been sponsored by Ministerio de Ciencia e Innovación (MINECO,
project BFU2011-23313), Junta de Extremadura and FEDER.
P57
PROBING THE KINETICS OF A MODEL HELICASE-NUCLEASE WITH
TEMPERATURE-CONTROLLED MAGNETIC TWEEZERS
Benjamin Gollnick1, Carolina Carrasco1, Francesca Zuttion1, Neville S. Gilhooly2,
Mark S. Dillingham2 and Fernando Moreno-Herrero1
Centro Nacional de Biotecnología, CSIC, Calle Darwin 3, Campus de Cantoblanco,
28049 Madrid, Spain, bgollnick@cnb.csic.es
2
DNA-Protein Interactions Unit, School of Biochemistry, Medical Sciences Building,
University of Bristol, University Walk, Bristol BS8 1TD, UK
1
Motor protein activities such as ATP hydrolysis and translocation are temperaturedependent; by studying their dynamics under different thermal conditions one can
estimate the associated physicochemical constants [1]. Here, we present a temperaturecontrolled Magnetic Tweezers setup designed for single-molecule experiments at up to
40 °C with 0.1 °C of precision. Its flexible heat control module can in principle adjust to
any surface-coupled optical microscopy method. Taking advantage of this instrument,
we have been able to compare translocation by individual copies of the bacterial DNA
helicase-nuclease complex AddAB [2-4] – an enzyme involved in the initial steps of
homologous double-stranded DNA break repair – at different thermal settings with
results inferred from bulk kinetics assays. Interestingly, although the two
complementary approaches give rise to a systematic difference between their
corresponding mean velocities measured at each temperature, they yield almost
identical estimates of the kinetic barrier of the translocation process, which turns out to
be around 21 kBT and hence similar to activation energies observed for other
translocating protein motors [5].
References
[1] Ralf Seidel, Joost G. P. Bloom, Cees Dekker and Mark D. Szczelkun, “Motor step size and ATP
coupling efficiency of the dsDNA translocase EcoR124I”, The EMBO Journal, vol. 27, no. 9, p.
1388-98, 2008.
[2] Joseph T. P. Yeeles, Emma J. Gwynn, Martin R. Webb and Mark S. Dillingham, “The AddAB
helicase-nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A
motor domain”, Nucleic Acids Research, vol. 39, no. 6, p. 2271-85, 2011.
[3] Joseph T. P. Yeeles, Kara van Aelst, Mark S. Dillingham and Fernando Moreno-Herrero,
“Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to
DNA unwinding by the AddAB helicase-nuclease”, Molecular Cell, vol. 42, no. 6, p. 806-16, 2011.
[4] Carolina Carrasco, Neville S. Gilhooly, Mark S. Dillingham and Fernando Moreno-Herrero, “On the
mechanism of recombination hotspot scanning during double-stranded DNA break resection”,
PNAS, vol. 110, no. 28, p. E2562-71, 2013.
[5] Aaron L. Lucius and Timothy M. Lohman, “Effects of temperature and ATP on the kinetic
mechanism and kinetic step-size for E. coli RecBCD helicase-catalyzed DNA unwinding”, Journal of
Molecular Biology, vol. 339, no. 4, p. 751-71, 2004.
P58
OPLS-AA parameters for 3-deoxy-D-manno-oct-2-ulosonyl acid disaccharide
and phosphorylated glucosamine disaccharide
Michał Markiewicz, Krzysztof Murzyn, Maciej Bratek and Marta Pasenkiewicz-Gierula
Department of Computational Biophysics and Bioinformatics, Jagiellonian University, Krakow, Poland
Optimized Potentials for Liquid Simulations (OPLS) is a set of potential energy
function parameters (forcefield) that have been successfully used in molecular
modelling studies of hydrated biomolecular systems. The OPLS forcefield has been
parameterized for various organic molecules including amino acids, nucleic acids, and
carbohydrates. Recent OPLS extensions contain new all-atoms parameters for
carbohydrates and lipids [1,2]. In the case of carbohydrates, OPLS has been developed
for monosaccharides only. In this presentation, preliminary results of the
parameterization of two disaccharides: di-3-deoxy-D-manno-oct-2-ulosonyl acid and
phosphorylated di-glucosamine, which constitute fragments of lipopolysaccharides in
the outer membrane of Gram-negative bacteria will be provided. Derived parameters
include point charges calculated with the GLYCAM06 charge fitting procedure for
conformers optimized at B3LYP6-31++G(d,p) level of theory and the glycosidic bond
rotational profile. Furthermore, results of conformer energy analysis for QM and MM
models will be presented.
Acknowledgement: This work is supported by the Polish National Science Center under grant no.
2011/01/B/NZ1/00081
References
[1] Kirschner, Karl N., et al. A glycam-based force field for simulations of lipopolysaccharide
membranes: parametrization and validation, Journal of Chemical Theory and Computation 8.11,
4719-4731, 2012.
[2] Murzyn, K., Bratek, M., and Pasenkiewicz-Gierula, M. Refined OPLS All-Atom Force Field
Parameters for n-Pentadecane, Methyl Acetate, and Dimethyl Phosphate, The Journal of Physical
Chemistry B, 117(51), 16388-16396, 2013.
P59
ORGANISATION OF INTERFACIAL REGIONS OF GLYCOLIPID BILAYERS
Marta Pasenkiewicz-Gierula, Krzysztof Baczyński and Krzysztof Murzyn
Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
The bilayer/water interface is one of three distinct zones of a lipid bilayer that strongly
determines the properties of the whole membrane. This is a polar, multi-component
region where polar and/or charged groups reside and generate long-range electrostatic
fields, and also participate in short-range interactions such as hydrogen bonds and
charge-charge interactions. In effect of these short-range interactions, intermolecular
lipid–lipid, lipid–water, and lipid–ion interactions are present at the interface, and they
depend on the chemical structures of the lipid molecules constituting the bilayer. In this
presentation, qualitative and quantitative analyses of short-range interactions at the
bilayer/water interface of three glycolipid bilayers are described. Two of these bilayers
constitute monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol
(DGDG), respectively, with -linolenoyl chains in both sn-2 and sn-3 positions; the
third one constitute Escherichia coli-specific hexa-acyl lipid-A (ECLA). The glycolipid
molecules used in the presented studies are hydrogen (H-) bond donors and acceptors,
and as such, are able to form intermolecular H-bonds both among themselves and with
water. Average numbers of direct and water mediated lipid-lipid H-bonds, and lipidwater H-bonds as well as the extent of networks of interlipid (direct and water
mediated) interactions are compared and related to the specificity of the chemical
structure of the lipid head groups.
Acknowledgements: This work was supported by grants 2011/01/B/NZ1/00081and N301 472638 of
the National Science Center, Poland. Some calculations were performed on the cluster purchased
under contract No. POIG.02.01.00-12-167/08, project MCB.
P60
PURIFICATION AND CHARACTERIZATION OF THE TRPV1 C-TERMINUS
Maite Artero1, Francisco Taberner2, Gregorio Fernández-Ballester2
and Antonio Ferrer-Montiel2,3
1
Institute of Molecular and Cellular Biology, Universidad Miguel Hernández, Elche (Alicante), Spain,
martero@umh.es
2
Institute of Molecular and Cellular Biology, Universidad Miguel Hernández, Elche (Alicante), Spain,
3
BIOFISIKA, The Basque Center for Biophysics, UPV/EHU-CSIC-FBB, Leioa (Bizkaia), Spain
Transient Receptor Potential Vallinoid subtype 1 (TRPV1) is a nonselective polymodal
ion channel that responds to both physical and chemical stimuli (e.g. voltage,
temperature, pH, capsaicin). It has been implicated in a wide variety of cellular and
physiological processes, including pain sensation. The active functional channel is a
tetramer. Each subunit contains six transmembrane segments (S1-S6), cytoplasmic Nand C- termini, and a pore-forming loop between the fifth and sixth transmembrane
domains. The C-terminal (682- 838aa) is involved in channel gating and its regulation.
The TRP domain, a highly conserved region in the N-end of the C-terminus (684721aa), is pivotal for subunit tetramerization. Furthermore, due to its proximity to the
channel gate, it is also a central molecular determinant of the channel allosteric
activation. Moreover, it contains modulatory domains able to bind Calmodulin,
phosphoinositides, as well as phosphorylation sites; and may contain part of the
temperature sensor. Consequently, disentangling the structure of the TRPV1 C-terminal
domain will contribute to increase our understanding of its implication in channel
function. Our aim was to produce pure, soluble and stable C- terminal protein for
structural analysis by either crystallography or NMR. For this task, we employed
different strategies including the use of: i) different solubility / affinity tags; ii) several
bacterial strains; iii) diverse experimental conditions (e.g. pH, salt concentration,
reductants); iv) various stabilizers of protein structure (Arginine, Sarcosine); and, v)
some proteins that may act as cytosolic chaperones (Calmodulin, Tubulin). We were
able to establish an experimental paradigm that produced purified, but partially stable,
C-terminal domain in solution. Preliminary biophysical characterization indicated that
the protein had a high tendency to aggregate in solution that prevented its full structural
investigation. A “divide and conquer”-wise approach is being implemented to gather
structural information on this key protein domain in TRPV1.
Acknowledgements: This work has been sponsored by Ministerio de Ciencia y Competitividad,
Consolider Ingenio 2010 and Generalitat Valenciana.
P61
MAGNETIC NANOPARTICLES AND THEIR INTERACTION WITH
CANCER CELLS ANALYZED BY INTEGRATIVE
MICROSCOPY AND SPECTROSCOPY
Francisco Javier Chichón1, José Javier Conesa1, Michele Chiappi2, Eva Pereiro2,
María Josefa Rodríguez1 and José L. Carrascosa1,3
Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049 Madrid, Spain.
2
ALBA Synchrotron Light Source. MISTRAL Beamline - Experiments division.
08290 Cerdanyola del Vallès, Barcelona, Spain.
3
Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia),
1
We have studied the interaction, internalization and accumulation of dimercaptosuccinic
acid-coated superparamagnetic iron oxide nanoparticles (DMSA-SPION), with average
diameter of 15 nm and negative surface charge, in MCF-7 breast cancer cells. Cell
cultures were incubated with 0.25 mg Fe ml-1 DMSA-SPIONs from 0 to 24 h.
Light microscopy, transmission electron microscopy (TEM) and Soft X-ray cryotomography were combined to characterize the interaction and accumulation of DMSASPION in MCF-7 cells, as well as the reorganization of the intracellular environment
due to the nanoparticle uptake. The correlation of these different techniques allowed to
visualize, at nanometric three-dimensional resolution, the whole cell without chemical
fixation or staining agents. Correlative microscopy was used to locate the cells
containing nanoparticles accumulated in endosomes. Reconstructed volumes show the
SPION-containing endosomal accumulation near the Golgi area, close to the nucleus.
Moreover, we acquired tilted series of the samples at different energies for tomographic
reconstructions. We used 700 eV (before the iron L3 edge) and 707 eV (on the L3 iron
edge) to localize in 3D the presence of iron. These series were aligned and reconstructed
to obtain the 3D distribution of iron particles within the sample.
Acknowledgements: These experiments were performed at Mistral beamline at ALBA Synchrotron
Light Facility with the collaboration of ALBA staff. We must acknowledge too Cedric Messaodi for
the software support from the Sergio Marco laboratory at Curie Institute.
P62
THE S6-TRPBOX LINKER DOMAIN IN TRPM8 PLAYS A CENTRAL ROLE
IN ALLOSTERIC CHANNEL ACTIVATION
Francisco José Taberner1, Ainara López-Córdoba1,2, Gregorio Fernández-Ballester1,
Yuri Korchev2 and Antonio Ferrer-Montiel1,3
1
Instituto de Biología Molecular y Celular. Universidad Miguel Hernández. Elche. Spain.
2
Imperial Collage School of Medicine, London, United Kingdom.
3
BIOFISIKA, the Basque Center for Biophysics, UPV/EHU-CSIC-FBB, Bilbao. Spain.
The ability of TRP channels to sense and respond to environmental and endogenous
cues is crucial in animal sensory physiology. However, the molecular mechanism of
channel gating is not well understood. The cytosolic C-terminus domain is considered to
be strategic in the channel opening. Recently, it has been pointed out that TRP box in
the C-terminus is pivotal for allosteric activation in this channel family. Here, we have
examined the role of the linker region between the TRPM8 inner gate and the TRP box
(referred to as the S6-TRPbox linker) to identify structural determinants of channel
gating. Using Step-wise substitutions of segments in the S6-TRPbox linker of TRPM8
channel with the cognate TRPV1 sequences, we identified Y981 and V986 as central
molecular determinants of channel function. Notably, mutation of these two positions in
a chimeric and wild type channels had a great impact on channel gating by voltage and
menthol, as evidenced by the modulation of the conductance-to-voltage (G-V)
relationships. Simulation of G-V curves using an allosteric model for channel activation
revealed that these mutations altered the allosteric constants that couple stimuli sensing
to pore opening. A molecular model of TRPM8, based on the recently reported TRPV1
structural model, showed that Y981 lies in a hydrophobic pocket at the end of the S6
transmembrane segment and is involved in inter-subunit interactions with residues from
neighbour subunits. V986 appears located in a tight hydrophobic cavity making intrasubunit interactions with residues of the S4-S5 linker. These findings substantiate a
gating mechanism whereby the TRP domain acts as a coupling domain for efficient
channel gating. Furthermore, they imply that protein-protein interactions of the TRP
domain may be targets for channel modulation and drug intervention.
P63
SINGLE-MOLECULE MECHANICAL CHARACTERIZATION OF
THE HmtSSB BINDING PROPERTIES TO ssDNA.
José A. Morin1, Laurie S. Kaguni2 and Borja Ibarra1
1
Imdea Nanociencia, Faraday, Nº 9 Ciudad Universitaria de Cantoblanco, 28049. Madrid (España)
2
Department of Biochemistry and Molecular biology, Michigan State University,
319 Biochemistry Building Michigan State University, East Lansing, MI 48824-1319, USA.
Mitochondrial DNA (mtDNA) encodes for most of the components of the cellular
electron transport chain. Mutations on mtDNA have been associated with respiratory
chain dysfunction, which in turn causes rare diseases in plants and humans. Therefore,
faithful replication of mtDNA is essential for the correct functioning of the cell. The
human mitochondrial single-stranded DNA binding protein (HmtSSB) plays a critical
role at the mtDNA replication fork, coordinating the interactions between singlestranded DNA (ssDNA), the DNA polymerase γ and the mtDNA helicase [1]. However,
little is known about the real time kinetics of the HmtSSB interaction with the ssDNA
and the structural and mechanical properties of the HmtSSB-coated ssDNA polymer;
which is essential information to understand the role of the HmtSSB on DNA
metabolism. Using optical tweezers ([2]) we have developed a single molecule
manipulation assay that addresses these questions. Our data show that the mechanical
properties of ssDNA change dramatically in the presence of HmtSSB and they strongly
depend on both the monovalent salt (0.05 to 0.3 M NaCl) and protein concentrations (5
to 200 nM) suggesting that HmtSSB interacts with ssDNA in two different modes [3].
Moreover, force pulling experiments designed specifically to test the stability of the
HmtSSB-coated polymer show that HmtSSB units associate with each other in a
cooperative fashion, whose strength also depends on ionic conditions and protein
concentration. Consistent with this observation, real time polymerization kinetics
exhibit two distinct behaviors, characterized by the apparent cooperativity, that
interchange continuously as the ratio of salt to protein concentration is tuned.
References
[1] M. Falkenberg, N. Larsson and C. Gustafsson, “DNA replication and transcription in mammalian
mitochondria.”, Annu Rev Biochem, 76, 679-699, 2007.
[2] S. B. Smith, Y. Cui and C. Bustamante, “Optical-trap force transducer that operates by direct
measurement of light momentum.”, Methods Enzymol, 361, 134-162, 2003.
[3] T. M. Lohman and M. E. Ferrari, “Eschericçhia coli single-stranded DNA-binding protein: multiple
DNA-binding modes and cooperativities.”, Annu Rev Biochem, 63, 527-570, 1994.
P64
NMR INSIGHTS INTO THE STRUCTURAL BASIS FOR ANTIMICROBIAL
ACTIVITY OF A SNAKE CATHELICIDIN PEPTIDE
Héctor Zamora1, Clara Pérez-Peinado2, Beatriz G. de la Torre2,
David Andreu2 and M. Ángeles Jiménez1
Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas (CSIC),
Serrano 119, 28006 Madrid, Spain.
majimenez@iqfr.csic.es
2
Department of Experimental and Health Sciences, Pompeu Fabra University,
Barcelona Biomedical Research Park, Dr. Aiguader 88, 08003 Barcelona, Spain.
1
Antimicrobial peptides (AMPs) constitute a promising alternative to address the
problem caused by the growing bacterial resistance to conventional antibiotic agents.
Usually cationic, AMPs naturally present in eukaryotic cells include α-helical peptides,
disulphide-rich β-hairpins, extended structures rich in particular amino acids such as
Gly, Pro, Arg, His or Trp, and even cyclic peptides. Despite such structural diversity,
they share interaction with bacterial membranes as an essential feature in their
mechanism of action. Therefore, experimental data about the structures of AMPs in
membrane environments and about AMP / membrane interactions are crucial to
understand their bactericidal activity at the molecular level. Nowadays, this is a field of
intense activity by applying both solid and solution NMR.
Here, we focus on a 34-residue antimicrobial cathelicidin like-peptide derived from the
South American Crotalus durissus rattle snake and apply solution 1H and 13C-NMR to
determine its three-dimensional structure in the presence of micelles. We have also
rationally dissected the 34-amino acid sequence into 14-mer N-terminal and 20-mer Cterminal peptides and examine their structural behavior in micellar media. For the three
peptides, this structural information is correlated with functional data on antimicrobial
activity against a panel of bacteria comprising both reference and clinical strains of
Gram-positive and Gram-negative species.
Acknowledgements: This work has been financially supported by Spanish MINECO (Project
number CTQ2011-22514).
P65
MODELLING THE INTERPLAY BETWEEN PROTEIN AND
LIPID AGGREGATION IN SUPPORTED MEMBRANES
Pablo González de Prado Salas1, Pedro Tarazona2 and Marisela Vélez3
Universidad Autónoma de Madrid, Madrid, Spain.
2
Centro de Investigación de Física de la Materia Condensada and Instituto de Ciencia de Materiales
Nicolás Cabrera, Universidad Autónoma de Madrid, Madrid, Spain
3
Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain
1
We present a theoretical model that deals with the complex interplay between lipid
segregation and the self-aggregation of lipid-attached proteins. The model, in contrast to
previous models that consider proteins only as passive elements affecting the lipid
distribution, describes the system including dynamic interactions between protein
monomers, the interactions between lipid components, and also a mixed term considering
both protein-lipid interactions.
This model is an extension of a previous one1,2 used to study the dynamics of the selfaggregating cytoskeletal protein FtsZ, and has been expanded to explain experimental
results performed on a well-defined system that includes two elements: FtsZ proteins and
a lipid bilayer with two lipid components. The model can reproduce the observed
differences determined by the orientation of the filaments on the surface, indicating that
all contributions to filament formation, including the interplay between torsion and an
specific anchoring, are needed to account for the observations. Furthermore, it points out
that lipid segregation can affect the length and curvature of protein filaments and that the
dynamic behaviour of the lipids and proteins might have different time scales, giving rise
to “memory effects”.
This simple model that considers a dynamic protein assembly on a fluid and active lipid
surface can be easily extended to other biologically relevant situations in which the
interplay between protein and lipid aggregation are both needed to fully describe the
system.
Acknowledgements: We acknowledge financial support by the Spanish Ministerio de Ciencia e Innovación
(Grant No. FIS2010-22047-C05), the Comunidad Autónoma de Madrid under program MODELICO
(Grant No. S2009/ESP-1691) and the Ministerio de Educación, Cultura y Deporte (FPU fellowship
program).
References
[1] Pablo González de Prado Salas, Mario Encinar, Marisela Vélez, Pedro Tarazona, “FtsZ protein on
bilayer membranes: Effects of specific lateral bonds”, Soft Matter, 9 (26), 6072–6079, 2013.
[2] Pablo González de Prado Salas, Ines Hörger, Fernando Martín-García, Jesús Mendieta, Álvaro Alonso,
Mario Encinar, Paulino Gómez-Puertas, Marisela Vélez, Pedro Tarazona, “Torsion and curvature of
FtsZ filaments”, Soft Matter, 10 (12), 1977–1986, 2014.
P66
APOCYNIN INHIBITS PURIFIED CYTOCHROME B5 REDUCTASE
ACTIVITY BY COMPETITION WITH NADH FOR ITS ACTIVE SITE
ON THE PROTEIN
Alejandro K. Samhan Arias1 and Carlos Gutiérrez-Merino
Dept. Biochemistry and Molecular Biology, Faculty of Sciences,
University of Extremadura, 06006-Badajoz, Spain
aksamhan@unex.es
Cytochrome b5 reductase (Cb5R) is a pleiotropic flavoprotein that catalyzes multiple one
electron reduction reactions in cells. In previous works, we have shown that Cb5R can
account for most of NADH dependent superoxide anion production of the neuronal
plasma membrane, being largely responsible of the superoxide anion overshot that is
observed during apoptosis of cerebellar granule neurons induced by extracellular
potassium deprivation [1]. In addition, NADPH oxidases have also been shown to be
sources of O2·- at the plasma membrane of cells under apoptotic stimulus in other type of
neurons [2]. Apocynin is one of the most frequently used inhibitors of NOX activity
inhibiting the plasma membrane NADPH oxidase activity of different cell types. We
found that 1mM of apocynin, a standard concentration used in culture to block NOX
activity, strongly inhibits the O2·- production by purified Cb5R, e.g. 79.7±13.7%
inhibition. Moreover, apocynin was also found to be a potent inhibitor of the NADH:
ferricyanide reductase activity of purified Cb5R, displaying an inhibition constant of 75
μM and reaching 90.1±10.4% inhibition with only 200 μM of apocynin. The analysis of
docking simulations suggested that apocynin inhibition of Cb5R activities is produced by
binding of this compound to the protein domain where the NADH binding site is located.
This hypothesis was experimentally assessed by kinetic analysis.
Acknowledgements
This work has been supported by Grant BFU2011-30178 of the Spanish Plan Nacional de I+D+I and by
Grant GR10092 of the Gobierno de Extremadura with FEDER co-financing.
References
[1] Samhan-Arias, A. K., Garcia-Bereguiain, M. A., Martin-Romero, F. J. and Gutierrez-Merino, C.
Clustering of plasma membrane-bound cytochrome b5 reductase within 'lipid raft' microdomains of
the neuronal plasma membrane. Mol. Cell. Neurosci. 40: 14-26; 2009.
[2] Sorce, S. and Krause, K. H. NOX enzymes in the central nervous system: from signaling to disease.
Antioxid. Redox Signal. 11: 2481-2504; 2009.
P67
FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF
PULMONARY SURFACTANT PROTEIN SP-C IN NANODISCS:
A NANOTECHNOLOGICAL APPROACH
Nuria Roldán1, Elena Aranda Serrano1, Jesús Pérez-Gil1 and Begoña García-Álvarez1
Department of Biochemistry and Molecular Biology I, Faculty of Biological Sciences,
Complutense University of Madrid
1
Because membrane proteins need to be taken out of their natural environment and
reconstituted into artificial milieus to perform structure-function studies, the search for
mimetic membranes that retain their native structure and functional activity constitutes a
challenge.
Nanodiscs emerge as a powerful tool for achieving this goal. These discoidal particles are
constituted by a phospholipid bilayer encircled by a membrane scaffold protein, which
determines the size of the nanodiscs. We are focused on the study of pulmonary surfactant
protein C (SP-C), a key component of the pulmonary surfactant lipoprotein complex that
reduces the surface tension in the pulmonary alveoli preventing them from collapse
during respiration. This transmembrane protein is a 35 amino acid peptide dually
palmitoylated in its N-terminal segment. SP-C is one of the most hydrophobic proteins
that are known and its low stability and the high tendency to form aggregated beta-sheets
in aqueous solvents difficult its structural and functional characterization even in the
presence of detergents.
In the present work, nanodiscs containing both native and a non-palmitoylated
recombinant version of SP-C have been obtained using POPC as a lipid membrane model.
The incorporation of SP-C into nanodiscs provides a novel approach for structural and
functional studies of this membrane protein in a bilayer mimetic system.
P68
A MULTI-TECHNIQUE BIOPHYSICAL APPROACH FOR
CHARACTERIZING OLIGOMERIZATION AND AGGREGATION OF
PROTEINS
Ana I. Díez, Vanesa Fernández Espín, Mar Collado-González,
José G. Hernández Cifre, José García de la Torre
Departamento de Química Física, Facultad de Química, Campus Regional de Excelencia
“Campus Mare Nostrum”, Universidad de Murcia, E-30071 Murcia, Spain.
Over the past few years, we have set up in our laboratory several techniques for
biophysical characterization of biomacromolecules in solution, including: (i) analytical
ultracentrifugation with both absorbance and interference detection, AUC/Abs-Int; (ii)
size-exclusion chromatography with multiple detection (light scattering and intrinsic
viscosity) SEC-LS/IV; (iii) dynamic light scattering and z-potential measurements
(DLSz). Collaborations with other institutions may extend the present potentiality as to
include other techniques like NMR relaxation and cryo-electron microscopy [1,2].
These techniques are complemented by (iv) our continuing work in the development of
tools for relating solution properties to the structure (size, shape and flexibility) of
biomolecules [1 – 4].
Thanks to their sensitivity, SEC-LS/IV and AUC/Abs-Inter are idoneous for the
characterization of oligomers or small-size aggregates. DLSz has less resolution, but
presents the great advantage its dynamic range, as it covers sizes from, roughly, 1 to
1000 nm, thus detecting large aggregates that are not detected by AUC or SEC/AbsInter, and with the added bonus of measuring the surface z-potential of proteins, which
is of evident importance in a variety of circumstances, ranging from protein association
to formation of protein crystals for crystallographic determinations. Our computational
tools can be employed to ascertain, say, the arrangement of subunits in homo- or heterooligomers, the influence of ordered or random oligomerization, or the overall shape and
eventual flexibility of large protein aggregates.
Examples of the information provided by these techniques, and their application to
various problems involving protein association and aggregation will be presented.
Acknowledgements: Supported by grants CTQ2012-33717 (Ministry of Economy and Competitiveness),
including FEDER funds, and 04486/GERM/06 (Fundación Seneca). A.I.D. is the recipient of a FPIMICINN grant.
References:
[1] J. García de la Torre, P. Bernadó, M. Pons. “Hydrodynamic models and computational methods for
NMR relaxation”, Methods in Enzymology, 349, 419-430 (2005)
[2] J. García de la Torre, O. Llorca, J.L. Carrascosa and J.M. Valpuesta. "HYDROMIC: Prediction of
hydrodynamic properties of rigid macromolecular structures obtained from electron microscopy".
European Biophysics Journal 30, 457-462 (2001).
[3] A. Ortega, D. Amorós, J. García de la Torre, “Prediction of hydrodynamic and other solution
properties of rigid proteins from atomic- and residue-level models” Biophysical Journal 101, 892-898
(2011)
[4] J. García de la Torre, Stephen E. Harding, “Hydrodynamic modelling of protein conformation in
solution: ELLIPS and HYDRO”, Biophysical Reviews, 5, 195-206 (2013).
P69
MOLECULAR STABILITY OF HUMAN ERYTHROCYTE INNER
MEMBRANE: PHASE DIAGRAM AND MECHANIC PROPERTIES
Silvia Isanta1, Iván López-Montero1, Ruddi Rodríguez-García1,2, Isabel Uruburu1,
and Francisco Monroy1,*
Mechanics of Biological Systems, Departamento de Química Física I,
Universidad Complutense de Madrid, 28040 Madrid, Spain.
*monroy@quim.ucm.es
1
IGDR, Faculté de Médecine, 2 avenue du Professeur Léon Bernard, CS 34317,
35043 Rennes Cedex, France.
2
The effect of the structure and lipid composition on membrane protein function could be
described in terms of multiple interactions, mainly electrostatic but highly specific like
hydrogen bonds and hydrophobic interactions. This complex game of interactions
between proteins and lipids of the bilayer leads to modifications on membrane structure,
as local curvature or hydration interactions with surrounding medium, forces
determining the activity of both proteins as the overall functionality of the membrane.
In this work we realized a study of bilayer stability constituted by lipids from inner
membrane of red blood cell belong the reconstitution in giant vesicles prepared from the
mixture of individual synthetic lipids. We have been studied the stability diagram over
the whole range of compositions of phosphatidylcholine (PC), phosphatidylserine (PS)
and phosphatidylethanolamine (PE), the three representative phospholipids of inner
membrane composition, mixed with different compositions of cholesterol. Determined
the stability region from lipid mixtures, it has been concluded about the stabilizing role
of cholesterol as well as the lack of stability of the native composition. Then, it has
explored the effect of incorporation of cytoskeleton proteins, which establish specific
interactions with lipids causing a net stabilization of the corresponding mixed
membranes [1]. This experimental evidence allows us to propose the lipid-protein
interaction as determinant of structural stabilization of native membrane. In addition,
studies of mechanics stability realized with fluctuation spectroscopy have corroborated
this conclusion, confirming the stabilizing effect of membrane protein. Finally, it is
proposed that these two aspects, structural and mechanic, are the responsible of an
adequate function of red blood cell membrane.
Acknowledgements: This work has been sponsored by Plan Nacional FIS2012-35723, ConsoliderIngenio 2010 en “Nanociencia Molecular” (CSD2007-0010), and S2009MAT-1507T (NOBIMAT).
Reference
[1] Iván López-Montero, Ruddi Rodríguez-García, and Francisco Monroy, “Artificial Spectrin Shells
Reconstituted on Giant Vesicles” J. Phys. Chem. Lett.2012, 3, 1583-1588.
P70
THREE-DIMENSIONAL STRUCTURE OF CbpL CHOLINE-BINDING
DOMAIN FROM Streptococcus pneumoniae AND COMPARED ANALYSIS
Javier Gutiérrez-Fernández1, Malek Saleh2, Sergio G. Bartual1, Thomas Pribyl2, Sven
Hammerschmidt2 and Juan A. Hermoso1
1
Department of Crystallography and Structural Biology, Institute of Physical-Chemistry “Rocasolano”,
Spanish National Research Council (CSIC), c/Serrano 119, Madrid, Spain. E-mail: xjavier@iqfr.csic.es
2
Department of Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics,
Ernst Moritz Arndt University of Greifswald, D-17487 Greifswald, Germany.
Choline binding proteins are a family of modulated proteins located in the gram positive
bacteria cell wall, showing a broad variety of activities. One of their modules, the so
called choline binding domain, is directly involved in the attachment of the cell wall.
The anchoring is formed directly with the peptidoglycan layer through cation-
interactions between repetitions of aromatic patches along the choline binding domain
and the choline moiety of teichoic and lipoteichoic acids present in the gram positive
cell wall.
In this work we have solved the three-dimensional structure of the choline binding
domain in CbpL, a choline binding protein from S. pneumoniae apparently involved in
adhesion to host cells during an infection. After cloning, expressing and purifying the
CbpL, we have obtained crystals in which only the CBD was present, probably due to a
proteolysis of the protein in its coiled regions. The overall structure shows the general
fold observed in other choline binding domains of the family. The CbpL choline
binding domain is formed by 9 repeats of two -strands each, giving a total of 9 choline
binding sites. These choline binding sites in CbpL can be divided into two groups,
namely, those that are formed by three aromatic residues and those that are formed by
four aromatic residues. They adopt a consecutive configuration along the CBD,
allowing the attachment to several lipoteichoic acids at the same time.
The new information obtained from the choline binding domain of CbpL can be used to
extend the knowledge about this kind of proteins. Due to the spatial organization of the
choline binding sites, the lipoteichoic acids could be arranged into a screw around the
choline binding domain or into a linear conformation along it. In addition, the
composition and configuration of the different types of choline binding sites could be
involved in the protein location in the cell wall with respect to other surface proteins.
Acknowledgements: This work was supported by grants from the Deutsche Forschungsgemeinschaft
DFG HA3125/4-2 (to SH), BFU2011-25326 (the Spanish Ministry of Economy and Competitiveness
to JAH), S2010/BMD-2457 (the Government of Community of Madrid to JAH) and EU FP7
CAREPNEUMO Grant EU-CP223111 from the European Union (to JAH and SH).
References
[1] I. Pérez-Dorado, S. Galan-Bartual, J. A. Hermoso, “Pneumococcal surface proteins: when the whole
is greater that the sum of its parts”. Mol. oral Microbiol. 27(4), 221-245, 2012.
[2] C. Frolet, M. Beniazza, L. Roux, B. Gallet, M. Noirclerc-Savoye, T. Vernet, A. M. Di Guilmi, “New
adhesin functions of surface-exposed pneumococcal proteins”. BMC Microbiol. 10,190, 2010.
[3] R. Hakenbeck, A. Madbour, D. Denapaite, R. Brückner, “Versatility of choline-binding proteins in
Streptococcus pneumoniae and commensal streptococci”. FEMS Microbiol. Rev., 33, 572-586,
2009.
P71
AN INVERTED HEXAGONAL PHASE (HII) OF
MONOGALACTOSYLDIACYLGLYCEROL (MGDG):
A MOLECULAR DYNAMICS STUDY.
Krzysztof Baczyń ki, Marta Pasenkiewicz-Gierula
Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, ul. Gronstajowa 7, 30-387 Kraków, Poland;
(krzysztof.baczynski@uj.edu.pl)
It is postulated that formation of an inverted hexagonal phase (HII) may occur in the
thylakoid membranes of chloroplasts. The occurrence of such a non-lamellar phase
could provide suitable environment for xanthophyll cycle. It has been shown that
solubility of pigments and efficiency of enzymes involved in this cycle is higher when
the process takes place in HII [1,2]. Monogalactosyldiacylglycerol (MGDG) is the most
abundant lipid component of the thylakoid membrane and due to its conical shape
promotes formation of HII. We constructed a computer model of the inverted hexagonal
phase of MGDG. The model was built of 360 MGDG and 10800 water molecules and
then equilibrated using the molecular dynamics simulation (MD) method. After 100-ns
of equilibration, the system was enlarged along the z-axis by adding to the original
system its copy. The enlarged system, consisting of 720 MGDG and 21600 water
molecules, remained stable during next 100 ns of MD simulation; its main structural
parameters: hexagonal spacing and radius of the water channel are analyzed and
compared with available experimental data.
Acknowledgements: This work is supported by the Polish National Science Center under grants
2011/01/B/NZ1/00081 and N301 472638.
References
[1] Goss, R.; Latowski, D.; Grzyb, J.; Vieler, A.; Lohr, M.; Wilhelm, C. & Strzalka, K. (2007), Lipid
dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems
resembling the lipid composition of the natural thylakoid membrane, Biochim. Biophys. Acta –
Biomemb. 1768, 67-75.
[2] Latowski, D.; Kruk, J.; Burda, K.; Skrzynecka-Jaskier, M.; Kostecka-Gugała, A. & Strzałka, K.
(2002), Kinetics of violaxanthin de-epoxidation by violaxanthin de-epoxidase, a xanthophyll cycle
enzyme, is regulated by membrane fluidity in model lipid bilayers, Eur. J. Biochem. 269, 4656-4665.
P72
FROM SENSING TO STABILIZATION OF MEMBRANE CURVATURE
BY HIV GAG
Artur Escalada1, Anna Snhyrova2, Hang Waters3, Joshua Zimmerberg3 and Vadim A. Frolov2,4
Biophysics Unit (CSIC, UPV/EHU), Department of Biochemistry and Molecular Biology,
University of the Basque Country and Fundación Biofísica Bizkaia, Leioa, Spain.
2
Biophysics Unit (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology,
University of the Basque Country, Leioa, Spain.
3
Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, MD 20892, USA.
4
IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
1
The geometry of HIV particles is supported by a protein shell formed by the polyprotein
Gag beneath the membrane of the virus envelope. Oligomerization of Gag on the inner
leaflet of the host plasma membrane is coupled to membrane curvature creation via yet
undetermined mechanism. Here we report that small oligomers of Gag discriminate
negative membrane curvature. Furthermore, the curvature intrinsic for viral particles
promote rapid and reversible condensation of Gag into fluid-like domains on the
membrane surface. With time, these domains acquire intrinsic spherical shape and
rigidity sufficient to support the shape. Theoretical analysis identifies condensationpolymerization mechanism as responsible for progressive conversion of the Gag activity
from curvature sensing to creation.
Acknowledgements:
Basque Government IT838-13
Basque Government Program Etortek IE12-332
P73
BIOLOGICAL INFORMATION OBTAINED BY SMALL ANGLE X-RAY
SCATTERING ON THE NON CRYSTALLINE DIFFRACTION (NCD)
BEAMLINE AT ALBA
Marc Malfois, Juan Carlos Martinez, Eva Crosas, Christina Kamma-Lorger
NCD beamline, ALBA Synchrotron Light Facility, Cerdanyola del Valles, Barcelona, Spain
mmalfois@cells.es
BioSAXS is a major experimental method to obtain very quickly low resolution
information about biological molecules. This method uses software package developed
at EMBL-Hamburg by D. Svergun group1. This technique is now well established and is
now used routinely on BioSAXS Beamlines. NCD is the Small Angle X-ray Scattering
beamline in the newly established Spanish synchrotron light facility, ALBA, situated in
Cerdanyola del Vallés in Barcelona. It allows the study of biological molecules like
proteins, DNA, protein-DNA complex etc… in condition very close to normal.
This technique combined with the ATSAS package provides:
 Ab initio low resolution envelop with dummy atoms2,3 or dummy residus4
 Modelling of multi-subunit complexes5
 Information about mixtures6
 Comparing the crystallographic structure with the Small Angle X-ray Scattering
(SAXS) data7
In conclusion, the NCD Beamline can solve low resolution shapes by using ATSAS that
is a versatile program suite that involves algorithms that can process various shapes of
proteins either single or multisubunit ones as well as protein-lipid or protein-DNA
clusters.
References:
[1]
[2]
[3]
[4]
[5]
[6]
[7]
P.V. Konarev, M.V. Petoukhov, V.V. Volkov, D.I. Svergun, “ATSAS 2.1, a program package for
small-angle scattering data analysis”, J. Appl. Cryst. 39, 277-286, 2006
D. Franke, D.I. Svergun, “DAMMIF, a program for rapid ab-initio shape determination in smallangle scattering”, J. Appl. Cryst., 42, 342-346, 2009
D. I. Svergun, “Restoring low resolution structure of biological macromolecules from solution
scattering using simulated annealing”, Biophys J., 2879-2886, 1999
D.I. Svergun, M.V. Petoukhov, M.H.J. Koch, “Determination of domain structure of proteins from
X-ray solution scattering”, Biophys. J., 80, 2946-2953, 2001.
M.V. Petoukhov, D.I. Svergun, “Global rigid body modelling of macromolecular complexes against
small-angle scattering data”, Biophys. J., 89, 1237-1250, 2005
P.V.Konarev, V.V.Volkov, A.V.Sokolova, M.H.J.Koch, D. I. Svergun, “PRIMUS - a Windows-PC
based system for small-angle scattering data analysis”, J Appl Cryst., 36, 1277-1282, 2003
D.I. Svergun, C. Barberato, M.H.J. Koch, “CRYSOL - a Program to Evaluate X-ray Solution
Scattering of Biological Macromolecules from Atomic Coordinates”, J. Appl. Cryst., 28, 768-773,
1995
P74
A HOMO-FRET-BASED METHOD FOR QUANTIFYING THE OLIGOMER
STOICHIOMETRY OF MEMBRANE-BOUND PROTEINS INVOLVED
IN A COOPERATIVE PARTITION EQUILIBRIUM
Ana M. Melo1, Aleksander Fedorov1, Manuel Prieto1 and Ana Coutinho1,2
CQFM and IN, Instituto Superior Técnico, Univ. de Lisboa, Lisboa, Portugal,
ana.coutinho@tecnico.ulisboa.pt
2
Dep. Química e Bioquímica, Faculdade de Ciências, Univ. de Lisboa, Lisboa, Portugal
1
The establishment of protein-protein interactions between membrane-bound proteins is
associated to several biological functions and dysfunctions. Here, we report an
analytical framework that uses energy homo transfer to directly probe quantitatively the
oligomerization state of membrane-bound proteins engaged in a three-state cooperative
partition [1]. Briefly, this model assumes that monomeric protein molecules partition
into the bilayer surface and reversibly assemble into oligomers with k subunits [2]. A
general equation relating the overall steady-state fluorescence anisotropy of the sample
to its fractional labeling was derived by considering explicitly that the anisotropy of
mixed oligomers containing i-labeled monomers is inversely proportional to the number
of labeled subunits per oligomer (Runnels and Scarlata limit) [3]. This method was very
robust in describing the electrostatic interaction of Alexa 488 fluorescently-labeled
lysozyme (Lz-A488) with phosphatidylserine-containing membranes. The pronounced
decrease detected in the fluorescence anisotropy of Lz-A488 always correlated with the
system reaching a high membrane surface density of the protein (at a low lipid-toprotein (L/P) molar ratio). The occurrence of energy homo transfer-induced
fluorescence depolarization was further confirmed by measuring the anisotropy decays
of Lz-A488 under these conditions. A global analysis of the steady-state anisotropy data
obtained under a wide range of experimental conditions (variable anionic lipid content
of the liposomes, L/P molar ratios and protein fractional labeling) confirmed that
membrane-bound Lz-A488 assembled into oligomeric complexes, possibly with a
stoichiometry of k= 6 ± 1. This study illustrates that even in the presence of a coupled
partition/oligomerization equilibria, steady-state anisotropy measurements provide a
simple and reliable tool to monitor the self-assembly of membrane-bound proteins.
Acknowledgements: A.M. Melo acknowledges the support of Fundação para a Ciência e Tecnologia
(FCT) via SFRH/BD/61723/2009. This work was supported by projects PTDC/QUIBIQ/099947/2008, PTDC/BBB-BQB/2661/2012 and RECI/CTM-POL/0342/2012 from FCT. A.M.
Melo current address is Dept Molecular Biophysics and Biochemistry, Yale University, New Haven,
Connecticut, United States.
References
[1] A.M. Melo, A. Fedorov, M. Prieto, A. Coutinho, Phys. Chem. Chem. Phys., 2014, DOI: 101039/c4cp00060a.
[2] A.M. Melo, J.C. Ricardo, A. Fedorov, M. Prieto, A. Coutinho, “Fluorescence detection of lipidinduced oligomeric intermediates involved in lysozyme "amyloid-like" fiber formation driven by
anionic membranes”, J. Phys. Chem. B, 117, 10, 2906-17, 2014.
[3] L.W. Runnels, S.F. Scarlata, “Theory and application of fluorescence homotransfer to melittin
oligomerization” Biophys. J., 69, 4,1569-8, 1995.
P75
BIOSAXS SOLUTION SCATTERING IN NCD BEAMLINE, ALBA:
A FAST METHOD FOR DETERMINING PROTEIN SHAPE IN SITU
Christina Kamma-Lorger, Juan Carlos Martinez, Eva Crosas, Marc Malfois
NCD beamline, ALBA Synchrotron Light Facility, Cerdanyola del Vallés, Barcelona, Spain
ckamma@cells.es
NCD is the small angle X-ray scattering beamline in the newly established Spanish
synchrotron light facility, ALBA, situated in Cerdanyola del Vallés in Barcelona. A major
experimental diffraction method that has been used widely in synchrotrons worldwide is
BioSAXS solution scattering that allows the study of proteins in solution. It is a rapid
technique and as proteins are in solution, they are being studied in conditions very close
to normal. In addition, by varying solution properties it is feasible to follow up in situ the
effect of changing conditions on protein structure. This technique is often used in
combination to others (i.e. NMR, AUC) and it provides very well defined information
regarding protein size and overall shape. The NCD beamline is now successfully
performing solution scattering experiments and soon there will also be a photon counting
detector that will provide even better data quality and much faster acquisitions in
following chemical processes. Data analysis packages are incorporated in the beamline,
with ATSAS1 (EMBL, Hamburg, Germany) being the most used one for solution
scattering experiments. Initially all data files are integrated in order to obtain I vs q, using
in house, NCD developed, software and subsequently, low resolution shape analysis of
proteins is performed using DAMMIN ab initio algorithm2 implemented in ATSAS.
Using the simulated annealing3 (SA) method and envelope functions additional ab initio
algorithms built a densely packed bead model of the protein with a diameter Dmax.
Several models are initially constructed and analyzed to finally select the average and the
most typical shape (Fig. 1).
Figure 1: Lysozyme ab initio model (© D. Svergun)
In conclusion, the NCD Beamline can solve low resolution shapes by using ATSAS, a
versatile program suite that involves algorithms that can process various shapes of
proteins either single or multisubunit ones as well as protein-lipid or protein-DNA
clusters.
References:
[1]
[2]
[3]
P.V. Konarev, , M.V. Petoukhov, V.V. Volkov, D.I. Svergun, “ ATSAS 2.1, a program package for
small-angle scattering data analysis”, J. Appl. Cryst., 39, 277-286, 2006.
D. Franke, D.I. Svergun, “DAMMIF, a program for rapid ab-initio shape determination in smallangle scattering”, J. Appl. Cryst., 42, 342-346, 2009.
D. I. Svergun, “Restoring low resolution structure of biological macromolecules from solution
scattering using simulated annealing”, Biophys J. 2879-2886, 1999.
P77
MECHANICAL CHARACTERIZATION OF TYPE 1
FIMBRIAL BACTERIAL ATTACHMENT PILI
Simon Poly1, Alvaro Alonso1 and Raul Pérez-Jiménez1
Group nanobiomechanics, CIC nanoGUNE Consolider, San Sebastián, Spain
1
Recently characterized type 1 pili are adhesive multisubunit fibers covering Gramnegative bacteria responsible for the adhesion of these ones to a large spectrum of
substrates [1]. Indeed, bacterial adhesion is mandatory to any form of surface
colonization and bacterial infection [2]. The pilus assembly is characterized by a
subunits sequential docking, with the assistance of a chaperone complex, and by the
stabilization of each subunits secondary structure through the exchange of one betasheet [3-4]. A detail analysis of each subunit secondary structure reveals that the only
contact between subunits is a shared triple interprotein beta-sheet structure [5]. The
reliance on hydrophobic interaction to sustain the important pulling forces, supported by
bacteria, hints at the complex mechanism implicated in this polyprotein structure. Using
collaboration of single-molecule in vitro measurement and in silico mechanical pulling
simulations, we have been able to analyses the mechanism implicated in gram-negative
bacterial attachment. Using molecular biology and Atomic Force Spectroscopy (AFS)
we were able to realize in vitro measurements that identified the forces and hinted at the
mechanism implicated in type 1 pili mechanical stability. AFS is a state-of-the-art
analysis technique developed in the last decade which has allowed investigating
mechanical properties at the single-molecule level. This analysis technique combined
with the development and production of circular permutant polyprotein allowed us to
study inter-protein contacts and shined a light at the mechanism implicated in the
attachment of gram-negative bacteria. Furthermore, we complemented our experimental
results by recreating, in silico, the subunits of the type 1 pili and generated simulations
that further demonstrate the mechanism of attachment taking place within the shared
triple interprotein beta-sheet structure. The elucidation of this attachment mechanism of
bacteria should lead us to the development of new therapeutics reducing the infection
efficiency of antibiotics-resistant strains.
References
[1] Geibel Sebastian, Procko Erik, Hultgren Scott J., Baker David, and Waksman Gabriel, “Structural
and energetic basis of folded-protein transport by the FimD usher”, Nature, no 496, pp. 243–246.
[2] Coutte Loïc, Alonso Sylvie, Reveneau Nathalie, Willery Eve, Quatannens Brigitte, Locht Camille,
and Jacob-Dubuisson Françoise, “Role of Adhesin Release for Mucosal Colonization by a Bacterial
Pathogen”, JEM, vol. 197, no. 6, pp. 735-742.
[3] Choudhury Devapriya, Thompson Andrew, Stojanoff Vivian, Langermann Solomon, Pinkner
Jerome, Hultgren Scott J., Knight Stefan D.,“X-ray Structure of the FimC-FimH Chaperone-Adhesin
Complex from Uropathogenic Escherichia coli”, Science, vol. 285, no. 5430, pp. 1061-1066.
[4] Puorger Chasper, Vetsch Michael, Wider Gerhard, Glockshuber Rudi, “Structure, folding and
stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli
strains”, J. Mol. Biol., vol. 412, no 3, pp. 520-535.
[5] RCSB – Protein Data Bank http://www.rcsb.org/pdb/explore/explore.do?structureId=4J3O
P78
FIREBALL/AMBER: A QM/MM METHOD FOR BIOMOLECULAR SYSTEMS
Jesús I. Mendieta-Moreno1,2,3, Ross C. Walker4, James P. Lewis5,
Paulino Gómez-Puertas2, Jesús Mendieta2,3 and José Ortega1
1
Departamento de Física Teórica de la Materia Condensada and Condensed Matter Physics Center
(IFIMAC), Universidad Autónoma de Madrid, ES-28049 Madrid, Spain
2
Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), ES-28049 Madrid, Spain
3
Biomol-Informatics SL, Campus UAM, ES-28049 Madrid, Spain
4
San Diego Supercomputer Center and Department of Chemistry and Biochemistry,
University of California-San Diego, La Jolla, California 92093, United States
5
West Virginia University, Morgantown, West Virginia 26506-6315, United States
In recent years, quantum mechanics/molecular mechanics (QM/MM) methods have
become an important computational tool for the study of chemical reactions and other
processes in biomolecular systems. Because of the complexity of biomolecules and the
desire to achieve converged sampling, it is important that the QM method presents a
good balance between accuracy and computational efficiency. Here, we report on the
implementation of a QM/MM technique that combines a DFT approach specially
designed for the study of complex systems using first-principles molecular dynamics
simulations (FIREBALL) with the AMBER force fields and simulation programs [1].
We also present the application of this method, using DFT QM/MM molecular
dynamics techniques, to study two different enzymatic reactions: phosphodiester bond
cleavage by RNase A and DNA polymerization by HIV reverse transcriptase. In
particular, the computational efficiency of our approach allowed the generation of freeenergy surface maps to explore the large conformational space for the reactions, thus
permitting a detailed analysis of alternative pathways.
Reference
[1] Jesús I. Mendieta-Moreno, Ross C. Walker, Paulino Gómez-Puertas, James P. Lewis, Jesús
Mendieta and José Ortega, “FIREBALL/AMBER: An efficient local-orbital DFT QM/MM method
for biomolecular systems”, Journal of Chemical Theory and Computation, DOI: 10.1021/ct500033w,
2014.
P79
LIPID MEMBRANE DOMAINS SHAPED BY ARTIFICIALLY
CHOLESTEROL RECYCLING IN MODEL VESICLES
Iván López-Montero1, Pilar Lillo2, and Francisco Monroy1
1
Departamento de Química Física I, Universidad Complutense, 28040 Madrid, Spain
2
Instituto de Química –Física Rocasolano, CSIC, 28006 Madrid, Spain
Lipid mixtures of phosphatidylcholine (PC), sphingomyelin (SM) and cholesterol (chol)
can lead to microscopic lipid phase separation on membrane model systems. This
observation suggests the existence of functional lipid rafts in living cells. However, cell
membranes are not static entities but affected by a continuous membrane recycling,
which could reduce the average domain radius in the tens of nm scale. Here, we study
the size of lipid domains in model vesicles under the action of cholesterol recycling. For
this purpose, lipid vesicles made of POPC/EggSM/Chol and DOPC/EggSM/Chol (at
5/2/3 and 3.5/4/2.5 molar ratios respectively) were fabricated in the presence of
progressively increasing concentrations of cholesterol-loaded and -unloaded
cyclodextrins (up to 50 M), able to uptake and release cholesterol molecules from/to
lipid bilayers. By means of time-resolved FRET performed on 100 nm size liposomes,
we have been able to measure a reduction of the lo-domain size up to 4% and 13 % for
POPC- and DOPC-containing bilayers respectively.
P80
QUANTAL KINETICS OF BAX-5 LEAKAGE IN SINGLE GUVS
ALLOWS COUNTING AND SIZING PORES
Edel Cunill1, Gustavo Fuertes², Orlando L. Sánchez Muñoz¹, Donata Janickaite¹ and Jesús
Salgado¹
E-mail: edel.cunill@uv.es
²Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL),
Heidelberg, Germany.
1
Bax-5 is a minimal active domain of the proapoptotic protein Bax. Like its full length
parent protein, this peptide displays strong pore-forming capacity in model membranes
[1]. Additionally, it can work as an independent inducer of apoptosis in cultured cells
and it has been proposed as a potential anticancer drug [2]. However, the structures and
formation mechanism of pores made by either full-length Bax or Bax-5 are unknown,
which hinders the development of therapeutic strategies based on pore-forming Bax
fragments. Here we report an approach which allows us describing individual pore
properties from the leakage kinetic of giant unilamellar vesicles (GUVs) studied by
fluorescence microscopy. Through the qualitative analysis of the kinetics of dye uptake
by single GUVs, we have previously detected changes in the size of pores due to
equilibration of Bax-5/membrane complexes [3]. We have now evaluated
quantitatively this kinetics for different populations of GUVs and determined the
number of pores per GUV and the distribution of area of individual pores. This analysis
stands on the quantal characteristics of the leakage kinetics, which manifests through
the discreteness of the distribution of total pore properties. By applying this method at
different times for a given GUV sample we are able to follow changes in the pore size
and the number of pores of particular GUVs. This results provide an unprecedented
dynamical view of the pore formation and evolution which can be extrapolated to the
Bax protein as well as to other amphipathic pore forming peptides.
References
[1] A.J. García-Sáez, M. Coraiola, M. Dalla Serra, I. Mingarro, G. Menestrina, J. Salgado, Peptides
derived from apoptotic Bax and Bid reproduce the poration activity of the parent full-length proteins,
Biophys J. 88 (2005) 3976–3990. doi:10.1529/biophysj.104.058008.
[2] J.G. Valero, L. Sancey, J. Kucharczak, Y. Guillemin, D. Gimenez, J. Prudent, et al., Bax-derived
membrane-active peptides act as potent and direct inducers of apoptosis in cancer cells, J Cell Sci.
124 (2011) 556–564. doi:10.1242/jcs.076745.
[3] G. Fuertes, A.J. García-Sáez, S. Esteban-Martín, D. Giménez, O.L. Sánchez-Muñoz, P. Schwille, et
al., Pores formed by Baxα5 relax to a smaller size and keep at equilibrium, Biophys J. 99 (2010)
2917–2925. doi:10.1016/j.bpj.2010.08.068.
P81
CONDENSATION OF DNA MEDIATED BY THE BACTERIAL
CENTROMERE BINDING PROTEIN Spo0J/ParB
César L. Pastrana1, James A. Taylor2, Mark S. Dillingham2
and Fernando Moreno-Herrero1
Department of Macromolecular Structures, Centro Nacional de Biotecnología,
Consejo Superior de Investigaciones Científicas, Madrid, Spain.
2
DNA:Protein Interactions Unit, School of Biochemistry, University of Bristol,
Bristol, United Kingdom.
1
The condensation and dynamic re-organization of the chromosome is crucial to the cell
cycle of all living organisms. In B. subtilis, the centromere binding protein Spo0J/ParB
has recently been implicated in the recruitment of condensins at parS sequences close to
the origin of replication [1, 2]. We have studied the binding of Spo0J to DNA molecules
using magnetic tweezers. At reduced forces, we have observed a progressive
condensation of the tethered DNA molecule in high Spo0J concentrations. Once the
condensation has been completed is possible to recover the original extension by
applying higher forces (4 pN). Our data shows that the condensation is a concentration
and force dependent process, with a stalling force at 3 pN, indicating that the
decondensed state can be restored by DNA processing machines [3].
The condensation phenomenon has been reproduced in torsionally and non-torsionally
constrained DNA, and in molecules both with and without parS sites, pointing to a nonspecific binding mode of Spo0J at high concentrations in agreement with bulk studies.
Using torsionally constrained molecules we have investigated structural implications of
the Spo0J binding on the DNA. We have determined a binding at forces higher than the
stalling force (4 pN), which triggers a decrease in the buckling number without the
induction of supercoiling. Nonetheless, the use of the novel technique freely orbiting
magnetic tweezers [4] has allowed us to track the torque executed by Spo0J during the
condensation, where the reduction in the extension is accompanied by a rotational
motion to a preferred direction.
Experiments with competitor DNA containing the parS sequence partially inhibited
condensation, revealed specificity for parS, and confirmed that the observed
condensation was Spo0J-mediated. Our data lead us to propose a model in which
condensation is induced by interactions between neighbouring Spo0J proteins due to the
Brownian motion of the DNA tether. Computer simulations based on this model
qualitatively reproduce our magnetic tweezers results.
References
1. Gruber S. and Errington, J. (2009) Recruitment of condensing to replication origin regions by
ParB/Spo0J promotes chromosome segregation in B.subtilis. Cell, 137: 685-696.
2. Sullivan, N.L., Marquis, K.A. and Rudner, D.Z. (2009) Recruitment of SMC by ParB-parS organizes
the origin region and promotes efficient chromosome segregation. Cell, 137: 697-707.
3. Wuite, G.J., Smith, S.B., Young, M., Keller, D. and Bustamante, C. (2000) Single-molecule studies of
the effect of template tension on T7 DNA polymerase activity. Nature 404: 103–6.
4. Lipfert, J., Wiggin, M., Kerssermakers, J.W.J, Pedaci, F. and Dekker, N.H. (2011) Freely orbiting
magnetic tweezers to directly monitor changes in the twist of nucleic acids. Nat. Commun., 2:439-448.
P82
STABILIZATION OF THE INTEGRIN β4-PLECTIN INTERACTION
WITH THIOL REACTIVE FLUORESCENT COMPOUNDS
José A. Manso1, Stephane Boivin2 and José M. de Pereda1
pereda@usal.es
2
European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Hamburg, Germany
1
Hemidesmosomes are junctional complexes that mediate the stable adhesion of
epithelial cells to the basement membrane (1-2). Integrin α6β4 and plectin are two
essential components of the hemidesmosomes. Plectin binds to the cytoplasmic domain
of the integrin β4 subunit; the primary interaction occurs between the actin-binding
domain (ABD) of plectin and a region of β4 consisting of two fibronectin type III
domains (FnIII-1,2) and a small sequence of the adjacent region termed the connecting
segment. We have previously elucidated the crystal structure of the primary α6β4plectin complex (3). Disruption of the integrin β4-plectin binding interface is linked to
the development of epidermolysis bullosa (EB), an inherited disease characterized by
fragility and blistering of the skin. Two missense mutations in β4, R1225H and
R1281W, inhibit the formation of the complex and have been detected in patients with
non-lethal form of EB with pyloric atresia (3-5). To date no compounds that modulate
the dynamics of hemidesmosomes are known. In this work, we have combined Cysscanning mutagenesis and labeling with thiol-reactive fluorescent compounds to explore
the stabilization of the β4-plectin interaction. Six plectin ABD mutants in which a Cys
was engineered near the β4-binding interface were created. The affinity for β4 of the
plectin mutants, each labeled with 9 different probes, was determined by fluorescence
assays. The binding of seven labeled-mutants of plectin to β4 was further characterized
by isothermal titration calorimetry. We have identified specific positions in plectin on
which some probes stabilize the integrin β4-plectin interaction. The largest stabilization
effect was observed when the plectin ABD was derivatized with probes that contain
naphthalene or pyrene groups. The labeling positions that stabilize the interaction are
located near a pocket at the rim of the β4-plectin interface. Stabilization of the β4plectin interaction rescued binding in the presence of the β4 mutations R1225H and
R1281W that are linked to the development of EB. Our results pave the way to search
for compounds that stabilize the hemidesmosomes. In addition, this approach could be
generally applied for probing other protein-protein interactions.
Acknowledgements: This work has been supported by the Junta de Castilla y León (grant CSI181A12 to
JMdP). The research leading to these results has received funding from the European Community's
Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). JAM
was recipient of a Postdoctoral Fellowship of the “Programa de Captación de Talento” (Botin
Foundation). We would like to extend special thanks to Rob Meijers and the Sample Preparation and
Characterization (SPC) facility at EMBL Hamburg for support and access to instruments.
References
[1] José M de Pereda, Esther Ortega, Noelia Alonso-García, María Gómez-Hernández, Arnoud
Sonnenberg, "Advances and perspectives of the architecture of hemidesmosomes", Cell Adh. Migr.,
3, 4, 361−363, 2009.
P82
[2] Sandy H.M. Litjens, José M de Pereda, Arnoud Sonnenberg, "Current insights into the formation
and breakdown of hemidesmosomes", Trends Cell Biol., 16, 7, 376−383, 2006.
[3] José M de Pereda, M Pilar Lillo, Arnoud Sonnenberg, "Structural basis of the interaction between
integrin α6β4 and plectin at the hemidesmosomes”, EMBO J., 28, 8, 1180−1190, 2009.
[4] Jan Koster, Ingrid Kuikman, Maaike Kreft, Arnoud Sonnenberg, "Two different mutations in the
cytoplasmic domain of the integrin β4 subunit in nonlethal forms of epidermolysis bullosa prevent
interaction of β4 with plectin", J. Invest. Dermatol., 117, 6, 1405-1411, 2001.
[5] Aoi Nakano, Leena Pulkkinen, Dedee Murrell, Joyce Rico, Anne W Lucky, Maria Garzon, Cathy A
Stevens, Stephen Robertson, Ellen Pfendner1, Jouni Uitto “Epidermolysis bullosa with congenital
pyloric atresia: novel mutations in the β4 integrin gene (ITGB4) and genotype/phenotype
correlations”, Pediatr. Res, 49, 618–626, 2001.
P83
EXPRESSION AND PURIFICATION OF MACROMOLECULAR
COMPLEXES ESSENTIAL FOR TELOMERASE MATURATION
Hugo Muñoz-Hernández1, Ángel Rivera-Calzada1 and Óscar Llorca1
Center for Biological Research (CIB), Spanish National Research Council (CSIC), Madrid, Spain,
hugomh@cib.csic.es
1
Telomeres correspond to specialized DNA-protein complexes that cap the end of
eukaryotic chromosomes and are essential for cellular and genomic stability.
Telomerase is the ribonucleoprotein responsible for maintaining a suitable length of
telomeres over extensive number of cell-division cycles. The telomerase holoenzyme
complex consists on a RNA component called TERC, the TERT protein responsible of
the reverse transcriptase activity of the holoenzyme, and an additional protein subunit
named dyskerin (1). Alterations and mutations in the telomerase components promote
the acquisition of tumoral phenotypes and ageing-associated diseases, as dyskeratosis
congenita. It has been described that pontin (also known as RuvBL1) and reptin
(RuvBL2), two ATPases of the AAA+ family, are essential for the assembly of a
functional telomerase holoenzyme (2). Our goal is to study the structural basis defining
the assembly and maturation process of telomerase and to describe the contributions of
pontin and reptin to these processes. To do this we are currently testing different
strategies for the expression and purification of truncated versions of dyskerin and the
recombinant human TERT. The purified components, together with pontin and reptin
whose purification has already been optimized by our group (3), will be used for in
vitro reconstitution of the different protein complexes corresponding to the different
steps of the telomerase maturation process. The assembled complexes will allow us to
unveil the functional, structural and mechanistic interactions that rule the maturation
process of the fundamental ribonucleoprotein telomerase.
Acknowledgements: This work is funded by the SAF2011-22988 project from the Spanish Ministry of
Economy and Competitiveness awarded to Prof. Oscar Llorca. Hugo Muñoz-Hernández is financed by
a PhD fellowship (FPI program, Spanish Ministry of Economy and Competitiveness).
References
1. Gardano, L., Holland, L., Oulton, R., Le Bihan, T., and Harrington, L. (2012). Native gel
electrophoresis of human telomerase distinguishes active complexes with or without dyskerin. Nucleic
Acids Res 40, e36.
2. Baek, S.H. (2008). When ATPases pontin and reptin met telomerase. Dev Cell 14, 459-461.
3. Lopez-Perrote, A., Munoz-Hernandez, H., Gil, D., and Llorca, O. (2012). Conformational transitions
regulate the exposure of a DNA-binding domain in the RuvBL1-RuvBL2 complex. Nucleic Acids Res
40(21):11086-99.
P84
STRUCTURE OF DIMERIC FRAGMENTS OF THE PLAKIN DOMAIN
Ana M Carballido1, José A Manso1, Rubén M. Buey1,2 and José M de Pereda1
ana.carballido@usal.es
2
Department of Microbiology and Genetics, University of Salamanca, Salamanca, Spain
1
The plakin family consists of very large proteins involved in the interconnection of
multiple systems of the cytoskeleton and their connection to adhesion complexes (1). In
mammals the plakin family includes plectin, desmoplakin, the bullous pemphigoid
antigen 1, the microtubule actin crosslinking factor 1, envoplakin, periplakin, and
epiplakin. Plakins have a tripartite modular structure. The N-terminal region contains a
conserved segment named the plakin domain (~1000 residues) that consists of up to
nine spectrin repeats (SR1-SR9) and an SH3 domain; the plakin domains mediate the
interaction with proteins in adhesion complexes (2). Downstream of the plakin domain
extends a central rod domain (900 to 1300 residues) that mediates homo-dimerization
via coiled-coil interactions. Finally, the C-terminal region contains binding sites for
intermediate filaments. We have recently solved the crystal structure of a 39-residues
fragment of the N-terminal region of the rod domain of plectin, which adopts a nonphysiological antiparallel coiled-coil in the crystal (3), suggesting that the formation of
parallel dimers requires larger segments of the rod domain. Here, we have created
dimeric fragments of the plakin domain of plectin and desmoplakin by exchanging the
large rod domain for the dimeric coiled-coil of GCN4 (4). These fragments were
analyzed by small angle x-ray scattering (SAXS) that confirmed the dimeric
oligomerization state. Analysis of the SAXS data indicates that the inter-protomer
contacts extend along the C-terminal segment of the plakin domain, but not in the Nterminal half of the plakin domain. These results have implications for the mechanical
properties of plakins and their interaction with other proteins in adhesion complexes.
Acknowledgements: This work has been supported by the Spanish Ministry of Economy and
Competitiveness (MINECO) and the European Regional Development Fund (grant BFU2012-32847).
The research leading to these results has received funding from the European Community's Seventh
Framework Program (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). AMC is recipient
of a FPI fellowship from the MINECO (BES-2010-038674). JAM was funded by a Postdoctoral
fellowship of the “Programa de Captación de Talento” (Botín Foundation). RMB is recipient of a contract
of the Ramon y Cajal program (MINECO).
References
[1] J-E. Bouameur, B. Favre, L. Borradori, “Plakins, a Versatile Family of Cytolinkers: Roles in Skin
Integrity and in Human Diseases”, J. Invest. Dermatol., 134, 4, 885-894, 2014.
[2] A. Sonnenberg , A.M. Rojas, J.M. de Pereda, “The structure of a tandem pair of spectrin repeats of
plectin reveals a modular organization of the plakin domain”, J. Mol. Biol, 368, 5, 1379-1391, 2007.
[3] M. Sammito, C. Millán, D.D. Rodríguez, I.M. de Ilarduya, K. Meindl, I. De Marino, G. Petrillo,
R.M. Buey, J.M. de Pereda, K. Zeth, G.M. Sheldrick, I. Usón, “Exploiting tertiary structure through
local folds for crystallographic phasing”, Nat. Methods, 10, 11, 1099-1101, 2013.
[4] E.K. O'Shea, J.D. Klemm, P.S. Kim, T. Alber, “X-ray structure of the GCN4 leucine zipper, a twostranded, parallel coiled coil”, Science, 254, 5031, 539-544, 1991.
P85
INTERDOMAIN FLEXIBILITY IN THE PLAKIN DOMAIN:
ANALYSIS BY SMALL ANGLE X-RAY SCATTERING
José A Manso1, Ana M Carballido1 and José M de Pereda1
jamanso@usal.es
1
Plakins are a family of large proteins that cross-link filament systems of the
cytoskeleton and tether them to membrane associated structures (1). Plakins are
essential for the integrity of tissues exposed to mechanical stress such as the skin and
muscle, and they are also important in the nervous system. Mammalian plakins include
plectin, desmoplakin, the bullous pemphigoid antigen 1 (BPAG1), the microtubule actin
crosslinking factor 1, envoplakin, periplakin, and epiplakin. Plakins are high molecular
weight proteins with a modular structure divided in three distinct regions, the N- and Cterminal regions harbor binding sites for other proteins and are separated by a central
rod domain. The N-terminal segment contains a region conserved in most plakins,
named the plakin domain, which is responsible for the subcellular localization of these
proteins. Plectin contains a complete plakin domain formed by an array of nine spectrin
repeats (SR1-SR9) and an SH3 domain inserted in the SR5 (2-3). Each SR is a three
helix bundle; adjacent SRs are connected by helical linkers and form elongated
structures. Nonetheless, the SR2 and SR3 are connected by a non-helical linker.
Upstream the SR1, plectin contains an actin binding domain (ABD) formed by two
calponin-homology domains. The ABD and the SR1 are connected by a short nonhelical linker. On the other hand, the epithelial variant of BPAG1 (BPAG1e) lacks the
ABD and the SR1. Here, we have used small angle x-ray scattering to analyze the
interdomain conformational variability in the N-terminal region of the plakin domains
of plectin and BPAG1e. The scattering data were analyzed using the ensemble
optimization method (4). Our results suggest that the N-terminal region of plakins
resemble articulated structures formed by apparently rigid segments connected by
flexible hinges.
Acknowledgements: This work has been supported by the Spanish Ministry of Economy and
Competitiveness (MINECO) and the European Regional Development Fund (grant BFU2012-32847).
The research leading to these results has received funding from the European Community's Seventh
Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). JAM was
recipient of a Postdoctoral Fellowship of the “Programa de Captación de Talento” (Botin Foundation).
AMC is recipient of a FPI fellowship from the MINECO (BES-2010-038674).
References
[1] J-E. Bouameur, B. Favre, L. Borradori, “Plakins, a Versatile Family of Cytolinkers: Roles in Skin
Integrity and in Human Diseases”, J. Invest. Dermatol., 134, 4, 885-894, 2014.
[2] A. Sonnenberg , A.M. Rojas, J.M. de Pereda, “The structure of a tandem pair of spectrin repeats of
plectin reveals a modular organization of the plakin domain”, J. Mol. Biol., 368, 5, 1379-1391,
2007.
[3] E. Ortega, R.M. Buey, A. Sonnenberg, J.M. de Pereda, “The structure of the plakin domain of plectin
reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat” J. Biol. Chem., 286,
14, 12429-12438, 2011.
[4] P. Bernado, E. Mylonas, M.V. Petoukhov, M. Blackledge, D.I. Svergun, “Structural
Characterization of Flexible Proteins Using Small-Angle X-ray Scattering”, J. Am. Chem. Soc., 129,
17, 5656-5664, 2007.
P86
MAGNETIC TWEEZERS STUDIES OF THE
TYPE IA TOPOISOMERASE RepC
Carolina Carrasco 1, César L. Pastrana 1, Parvez Akhtar 2, Sanford H. Leuba 2,
Saleem A. Khan 2, and Fernando Moreno-Herrero1
1
Centro Nacional de Biotecnología, CSIC, Campus UAM, Darwin 3, 28049 Cantoblanco, Madrid, Spain
2
Dept. of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, EEUU
A precise and time-dependent control of the level of DNA supercoiling is essential in
many cellular processes such as replication, recombination and transcription. This task
is carried out by the action of topoisomerases by introducing transient single or doublestrand breaks in the DNA [1]. We have used Magnetic Tweezers (MT) to study the
enzymatic activity of RepC, a plasmid-encoded type IA topoisomerase from
Staphylococcus aureus [2]. SaRepC is the replication initiator protein of plasmid pT181
and constitutes a prototype to study rolling-circle replication (RCR) of plasmids in
Gram-positive bacteria. RepC nicks plasmid DNA at the origin of replication and
remains covalently bound to the DNA through a phosphotyrosine bond while supercoils
are released. Then, RepC ligates the previously formed nick and releases the relaxed
DNA [3]. In MT, a single molecule of DNA is tethered between a micrometer-size
magnetic bead and the surface of a liquid cell. By rotating a pair of magnets above the
liquid cell, different number of supercoils can be induced on torsionally-constrained
DNA molecules [4]. We measured both nicking and re-ligation activities of RepC on
supercoiled DNA containing its nick site. RepC was only active on negatively
supercoiled DNA suggesting that binding to DNA requires the formation of specific
DNA structures at the origin region. Moreover, supercoils were released in a vast
majority of cases in a single step. Characterization of RepC activity constitutes a
starting point to study the combined activities of topoisomerases and helicases in RCR
of plasmids.
References
[1] Champoux, J. J. (2001). DNA Topoisomerases: Structure, Function, and Mechanism. Annu Rev
Biochem 70, 635-92.
[2] Koepsel, R. R., et al. (1985). The replication initiator protein of plasmid pTl81 has sequence-specific
endonuclease and topoisomerase-like activities. Proc. Natl. Acad. Sci. USA 82, 6845-6849.
[3] Koepsel, R. R., et al. (1986). Sequence-specific interaction between the replication initiator protein of
plasmid pT181 and its origin of replication. Proc. Natl. Acad. Sci. USA 83, 5484-5488.
[4] Strick T. R., et al. (1998). Behavior of supercoiled DNA. Biophy Journal74, 2016–2028.
P87
CONFORMATIONAL CHANGES LEADING TO DNA DELIVERY IN T7
BACTERIOPHAGE UPON RECEPTOR INTERACTION
Ana Cuervo1, Verónica González-García1, Mar Pulido-Cid1, Mónica Chagoyen2, Rocío Arranz1,
José R. Castón1, José J. Fernández1, Carmela García-Doval1, 3, José M. Valpuesta1,
Mark J. van Raaij1, Jaime Martín-Benito1 and José L. Carrascosa1, 4
1
Structure of Macromolecules Department Centro Nacional de Biotecnología, CSIC. Darwin 3,
2
Systems Biology Department, Centro Nacional de Biotecnología, CSIC. Darwin 3,
3
Current address, Department of Biochemistry, University of Zurich, CH-8057 Zurich,
Switzerland.
4
Unidad de NanoBiotecnologia, Instituto Madrileño de Estudios Avanzados en Nanociencia
(IMDEA Nanociencia). Cantoblanco, 28049 Madrid, Spain.
The bacterial envelope is the major barrier that bacteriophages have to overcome to
successfully deliver their genome inside the cytoplasm, and the mechanism involved in
the transport of the viral genome is one of the most intriguing processes in the bacterial
virus biology. Most of bacteriophages use a specialised complex (called tail) to deliver
its genome without disrupting cellular integrity. T7 bacteriophage is a wellcharacterized member of the Podoviridae bacteriophage family infecting E. coli, and it
presents a short non-contractile tail that assembles sequentially in the viral head after
DNA packaging [1]. T7 tail is a complex composed by a tubular structure surrounded
by fibers with a central channel serves as a conduit for DNA ejection. Despite their
structural simplicity, Podoviridae tail machines follow a complex mechanism of
ejection. Their short length forces them to recruit additional internal proteins to form a
channel trough the bacterial membrane. Low-resolution studies using cryo-ET have
permitted to propose the main steps of the T7 infection mechanism [2], but the lack of
knowledge of the receptor for these viruses has prevented until now to characterize the
conformational changes at a higher resolution. In this work, we identified the protein
and lipid bacterial compounds used as a receptor for T7. Our results shows that porins
OmpA and OmpF help to the viral adsorption, but the presence of rough LPS from E.
coli is sufficient to trigger the complete DNA delivery in vitro. These findings permitted
us to set up an in vitro ejection system for T7 bacteriophage to analyze by cryo-EM and
single particle reconstruction methods the structure of the tail after ejection. The
comparison of the pre and post-ejected conformations allowed us to depict the
conformational changes that take place in the tail complex during DNA delivery, and to
propose a model to describe the first steps of the T7 infection mechanism.
Acknowledgements: This work was supported by grants BFU2011-29038 (to JLC), BFU2011-25090
(to JM-B) and BFU2011-24843 (to MJvanRj) from the Ministry of Economy and Competitiveness.
References
[1] Cuervo, A., Pulido-Cid, M., Chagoyen, M., Arranz, R., Gonzalez-Garcia, V.A., Garcia-Doval, C.,
Caston, J.R., Valpuesta, J.M., van Raaij, M.J., Martin-Benito, J. and Carrascosa J.L. “Structural
characterization of the bacteriophage T7 tail machinery”,The Journal of biological chemistry, 288,
26290-26299, 2013.
[2] Hu, B., Margolin, W., Molineux, I.J., and Liu, J. “The bacteriophage T7 virion undergoes extensive
structural remodeling during infection”, Science, 339, 576-579, 2013.
P88
PORES OF MELITTIN IN SUPPORTED LIPID MONOLAYERS
AS OBSERVED BY AFM
Diana Giménez, Orlando L. Sánchez Muñoz and Jesús Salgado
E-mail: jesus.salgado@uv.es
Melittin, a membrane lytic peptide from bee venom is often considered archetypal
among the large and diverse number of pore-foming peptides. It is used as a model for
the understanding of the structure and mechanism of these systems and as a scaffold for
the development of new pore-forming molecules with potential uses in medicine and
biotechnology [1]. However, the structure of melittin in the pores is still unknown and
there is even debate about the existence of these pores and their importance for melittin
function [1,2]. In this work we report the direct observation of melittin-induced pores in
supported lipid monolayers by atomic force microscopy (AFM). Because the interaction
of melittin with lipid bilayers occurs essentially at the interface level, the monolayer
provides an analogous docking site for the peptide. It is therefore a good model for the
initial stages of binding to membranes. We will show that the monolayer systems can
also be considered adequate for trapping relevant structures of the peptide-membrane
complex, including pores. Moreover, the monolayer may represent a way to shift the
thermodynamic balance between melittin bound species and increase the occurrence of
pores compared to their abundance in bilayers. We describe the nanometer scale
properties of these pores, including a rim of protruding material which is likely
associated to the presence of melittin molecules. This study may represent a significant
step forward towards the structural investigation of dynamic pore forming systems
using monolayers.
References
[1] A.J. Krauson, J. He, W.C. Wimley, “Gain-of-Function Analogues of the Pore-Forming Peptide
Melittin Selected by Orthogonal High-Throughput Screening”, J. Am. Chem. Soc, 134, 12732–
12741 (2012). doi:10.1021/ja3042004.
[2] M.-T. Lee, T.-L. Sun, W.-C. Hung, H.W. Huang, “Process of inducing pores in membranes by
melittin”, Proc. Natl. Acad. Sci. 110, 14243–14248 (2013). doi:10.1073/pnas.1307010110.
P89
STRUCTURE-AFFINITY RELATIONSHIP (SAFIR) FOR THE mRNA CAP
ANALOGS BINDING TO C. elegans DcpS ENZYME
Anna Wypijewska del Nogal1, Marius D. Surleac2, Joanna Kowalska1,
Maciej Lukaszewicz1, Jacek Jemielity1,3, Martin Bisaillon4,
Edward Darzynkiewicz1,3, Adina L. Milac2 and Elzbieta Bojarska1,3
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw,
Warsaw, Poland, wypijewska.del.nogal@gmail.com
2
Department of Bioinformatics and Structural Biochemistry,
Institute of Biochemistry of the Romanian Academy, Bucharest, Romania
3
Centre of New Technologies, University of Warsaw, Warsaw, Poland
4
Department of Biochemistry, University of Sherbrooke, Sherbrooke, Quebec, Canada
1
DcpS (Decapping Scavenger) enzyme proceeds degradation of the 5’ end of mRNAs,
referred as cap, when it is released from mRNA body by other enzymes. Cap consists of
7-methylguanosine (m7G) and a triphosphate chain (ppp). The specificity of DcpS is the
highest for the cap-containing dinucleotides (m7GpppN, where N – any nucleoside).
The hydrolysis products are 7-methyloguanosine monophosphate (m7Gp) and a
nucleoside diphosphate (ppN). The hydrolytic activity of DcpS towards free caps
prevents inhibition of other cap-binding proteins, such as CBC, eIF4E or Dcp2.
Notably, these proteins trigger crucial processes in gene expression: splicing, translation
initiation and mRNA degradation, respectively. Moreover, DcpS has been proposed as a
therapeutic target for spinal muscular atrophy (SMA) – an incurable disease, which
leads to disorders in muscle movement in less severe types, and infant death in the most
severe type.
Here we determine binding affinity of several mono- and dinucleotide cap
analogs for DcpS from the C. elegans model organism. Modifications of the cap
structure were introduced into the phosphate chain (hydrolysis site), in order to obtain
caps resistant to DcpS and suitable for binding affinity studies. Several types of
modifications were investigated, either bridging (with CH2 or NH substituents) or nonbridging (with S substituent), as well as caps of various length of the phosphate chain
(from one to four phosphate groups). Employing fluorescence titration, we determined
the equilibrium association constants (KAS) and Gibbs free energies of binding (G0).
Structure-affinity relationship (SAFIR) analysis of the cap analogs enabled us to
identify the functional groups of the cap important for the efficient DcpS-cap complex
formation. The subsequent computational docking of cap analogs into the DcpS enzyme
active site let us to pinpoint how phosphate chain mobility and orientation in the capbinding pocket depend on the number of phosphates, the substituent type and the
presence of the second nucleoside.
The comparison of C. elegans DcpS with its well known human homolog
provides general insight into mechanism of DcpS catalysis. Our results might be useful
for finding DcpS inhibitors of potential therapeutic value, e.g., in SMA, and for
examining the consequences of DcpS inhibition in a model organism.
P90
ROLE OF THE P75 TRANSMEMBRANE DOMAIN DIMERIZATION
IN RECEPTOR ACTIVATION AND FUNCTION
Irmina García-Carpio1, Kirill D. Nadezhdin2,, Alexander S. Arseniev2
and Marçal Vilar1
1
2
Neurodegeneration Unit. UFIEC-ISCIII, Madrid, Spain.
Icarpio@isciii.es
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences,
Moscow 117997, Russian Federation.
p75 neurotrophin receptor (p75 NTR), a member of the Tumor Necrosis Factor
superfamily is best known for its role in mediating neuron cell death during
development or after injury but it also regulates cell proliferation, axon guidance or
survival. The key to understand its signaling could rely in its structure and
conformational states. It has been described that p75 forms disulphide-linked dimers
through the Cys257 in the transmembrane domain and that it is essential for its NGF
mediated signaling1. Using ToxRED here we demonstrate that p75 use two different
interfaces of dimerization; one more stable promoted by Cys257 dimerization and the
other by a motif of the form AxxxG. Both dimers are in equilibrium unless the disulfide
bond is formed. Analysis of different mutations, in a mammalian cell viability assay,
allows us to determine the role played by both dimerization interfaces in p75 function.
Our functional data reveals the key role that plays the Cys257 in p75 dimer assembly,
redistribution and activation of the receptor in vivo.
Acknowledgements: This work has been sponsored by MINECO BFU2010-15276
Reference
[1] Vilar, M., Charalampopoulos, I., Kenchappa, R. S., Simi, A., Karaca, E., Reversi, A., Choi, S.,
Bothwell, M., Mingarro, I., Friedman, W. J., Schiavo,G., Bastiaens, P. I., Verveer, P. J., Carter, B.
D., and Ibáñez, C. F. Activation of the p75 neurotrophin receptor through conformational
rearrangement of disulfide-linked receptor dimers. Neuron 62, 72–83, (2009)
P91
N-TERMINAL ACETYLATION OF -SYNUCLEIN REDUCES THE
POPULATION OF PARTIALLY FOLDED OLIGOMERS AND
INHIBITS AMYLOID AGGREGATION INDUCED BY SDS
David Ruzafa, Bertrand Morel and Francisco Conejero-Lara
Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences,
University of Granada, 18071 Granada, Spain
ruzafa@ugr.es
The Parkinson’s disease (PD) is the most common neurodegenerative motor system
disorder affecting more than 1% of the population over 65. PD is characterized by the
loss of dopaminergic neurons in the “substantia nigra” and the appearance of
intraneuronal inclusions, known as Lewy bodies. The major components of the Lewy
bodies are fibrillar aggregates of -Synuclein (-Syn) and its oligomeric forms. -Syn
oligomerization is believed to play a key role in the progress of PD 1.
Although -Syn has always been described as an intrinsically disordered protein, it
adopts -helical structures when it binds to negatively charged membranes 2 inducing
curvature in them. This has suggested a membrane remodeling function for -Syn.
Recent evidence suggests that unfolded -Syn monomers exists under physiological
conditions in equilibrium with oligomeric forms. Moreover, this equilibrium appears to
be modified by N-terminal acetylation 3. This posttranscriptional modification increases
the hydrophobicity of the N-terminal region, stabilizes its -helical structure, enhances
the affinity with anionic lipid surfaces and somehow decreases the aggregation
propensity4. However, the underlying mechanisms of these effects remain unclear.
Sodium dodecyl sulfate (SDS) is an anionic surfactant commonly used in biophysical
studies to mimic membrane environments for proteins and it has been extensively used
with -Syn 5,6. Low SDS concentrations (0.5-2 mM) stabilize oligomeric and partially
folded states and increase amyloid fibrillation, whereas higher SDS concentrations
decrease this amyloidogenic propensity with higher -helix content.
In this work, we compare the formation of partially folded oligomers of N-acetylated
and unacetylated -Syn in presence of small concentrations of SDS and explain the
different behavior related to their amyloidogenic propensity. We find that the SDSassociated oligomers constitute optimal species for spontaneous and efficient formation
of amyloid nuclei, which further drive lag-free amyloid fibrillation. SDS-induced -Syn
fibrillation kinetics could be amenable to a quantitative analysis using our previously
developed nucleation model 7.
Acknowledgements: This work has been sponsored by the Spanish Ministry of Science and
Innovation (grant BIO2009-07317), and the European Regional Development Fund of the
European Union.
(1) Lorenzen, N. et al. The Role of Stable -Synuclein Oligomers in the Molecular Events Underlying Amyloid Formation. Journal
of the American Chemical Society 2014, 136, 3859-68,.
(2) Trexler, A. J.; Rhoades, E. -synuclein binds large unilamellar vesicles as an extended helix. Biochemistry 2009, 48, 2304-6.
(3) Bartels, T.; Choi, J. G.; Selkoe, D. J. -Synuclein occurs physiologically as a helically folded tetramer that resists aggregation.
Nature 2011, 477, 107-U123.
(4) Kang, L. et al. J. N-terminal acetylation of -synuclein induces increased transient helical propensity and decreased aggregation
rates in the intrinsically disordered monomer. Protein Science 2012, 21, 911-917.
(5) Ahmad, M. F.; Ramakrishna, T.; Raman, B.; Rao Ch, M. Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human synuclein. Journal of molecular biology 2006, 364, 1061-1072.
(6) Giehm, L.; Oliveira, C. L.; Christiansen, G.; Pedersen, J. S.; Otzen, D. E. SDS-induced fibrillation of alpha-synuclein: an
alternative fibrillation pathway. Journal of Molecular Biology 2010, 401, 115-133.
(7) Ruzafa, D.; Morel, B.; Varela, L.; Azuaga, A. I.; Conejero-Lara, F. Characterization of oligomers of heterogeneous size as
precursors of amyloid fibril nucleation of an SH3 domain: an experimental kinetics study. PloS one 2012, 7, e49690.
P92
ROLE OF WW DOMAINS IN HUMAN YAP ISOFORMS
Pedro Buzón1, Manuel Iglesias‐Bexiga1, Francisco Castillo1, Eva S. Cobos1, Tsutomu Oka2,
Marius Sudol2 and Irene Luque1,*
Department of Physical Chemistry and Institute of Biotechnology, Faculty of Sciences,
University of Granada, 18071 Granada, Spain
2
Weis Center for Research, Geisinger Clinic, M.C. 26‐08, 100 North Academy Avenue,
Danville, PA 17822‐2608, USA
1
YAP, Yes kinase associated protein, is a WW domain‐containing effector of the Hippo
tumour suppressor pathway, and the object of a great interest as a potent oncogene and
stemness factor. We present here a detailed study of YAP binding, focused on its WW
domains, including phage‐display techniques combined with in vivo functional studies
and a detailed biophysical characterization of the conformational equilibrium and
binding preferences of the hYAP‐WW1, hYAP‐WW2 and hYAP‐WW1‐WW2 tandem.
Functional studies with PATCHED1 (PTCH1) peptide, selected from the set of
strongest binders, show that YAP and PTCH1 form a complex and signal together,
providing the first molecular link between YAP and a member of the Sonic Hedgehog
pathway. Also, our results reveal that the WW domains in hYAP behave as two
independent units with different binding preferences and different levels of regulation
by tyrosine phosphorylation of the PPxY core. A detailed thermodynamic analysis of
the interactions of a set of functionally relevant peptides, including ligands derived from
PTCH1, and non-natural binders obtained by phage‐display, like Pd4 peptide, indicates
that electrostatic interactions play a critical role in the determination of binding
specificities between Yap WW domains. To improve our knowledge about WW domain
specificity, structural determination of WW2‐Pd4 and WWtandem‐Pd4 complex by X‐
ray crystallography will be part of our future perspectives. The relevance of these results
for the understanding of the molecular function of different YAP isoforms and its role
in network signalling will be discussed.
P93
INFLUENCE OF IONIC STRENGTH ON THE FLEXIBILITY OF ALGINATE
STUDIED BY SIZE EXCLUSION CHROMATOGRAPHY
M. Mar Collado-González, Vanesa Fernández Espín, F. Guillermo Díaz Baños,
José García de la Torre
Department of Physical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence
“Campus Mare Nostrum”. University of Murcia, 30071 Murcia, Spain
In this work we present a methodology based in experimental measurements from SEC
chromatography and the use of the in-house software HYDFIT to characterize flexibility of
biopolymers. In this case, flexibility of alginate has been studied.
Alginate is a structural non branched polyelectrolytic biopolymer present in brown seaweeds
(Laminaria sp., Macrocystis sp., among others) with numerous applications in biomedical
science. Its chemical structure consists in β(1→4)-D-manuronic acid linked to C5-epimer,
α(1→4)-L-guluronic acid. In addition to the distribution of these monomers, the
polyelectrolytic nature of alginate are essential to explain the flexibility of this biopolymer.
That is the reason why the ionic strength of the solution has a strong influence in the flexibility
of this macromolecule.
Two different assumptions concerning mass per unit of length lead to different conclusions.
First: persistence length decreases with ionic strength (the intrinsic component of the
persistence length is 11.3 nm and the electrostatic component is 6 nm when ionic strength is
0.01). Second: persistence length is independent of ionic strength (12 nm). Either of these
options shows that the wormlike model in itself is not sufficient to explain flexibility over the
whole range of chain lengths for these polyelectrolytes. A plausible explanation could be the
presence of a combination of short-range and long-range screening effects of the ions of the
solutions. This would also explain some data found in the literature regarding alginate
flexibility.
Acknowledgments
This work was performed within a Grupo de Excelencia de la Región de Murcia (grant 04531/GERM/06).
Support also provided by Grant CTQ-2012-33717 from Ministerio de Economía y Competitividad
including FEDER funds.
P94
SINGLE MOLECULE FRET REVEALS DYNAMIC OLIGOMERS
IN AN AMYLOIDOGENIC SH3 DOMAIN
Fabio Castello,1 Salvador Casares2 and Angel Orte1,*
1
Department of Physical Chemistry. Faculty of Pharmacy. Campus Cartuja, 18071 Granada (Spain);
E-Mail: fabiocastello@ugr.es (F.C.)
2
Department of Physical Chemistry. Faculty of Sciences. Campus Fuentenueva,
18071 Granada (Spain); E-Mail: scasares@ugr.es (S.C.)
* Corresponding author e-Mail: angelort@ugr.es
Tel.: +34-958-243825; Fax: +34-958-244090.
The structural and dynamic characterization of the on-pathway intermediates involved in
the mechanism of amyloid fibril formation, one of the major biomedical challenges of our
time, remains to be determined. In addition to mature fibrils, there are different oligomeric
structures implicated in the rate-limiting step of the nucleation process. Nucleation plays
a central role in the conversion of oligomers to protofibrils and mature fibrils. These
oligomers are associated with the neuronal toxicity of amyloids deposition.
Distinguishing those soluble intermediate oligomers can be achieved using singlemolecule fluorescence (SMF) to study the process at the molecular level. Counting
molecules one by one by SMF techniques provides valuable information on
heterogeneous systems. This kind of information would be hard or impossible to obtain
from conventional, bulk methodologies.
We focused on the formation of the early oligomeric aggregates of the highly
amyloidogenic N47A mutant of the alpha spectrin SH3 domain as a model. We labeled
the SH3 monomers with either a donor (Atto® 488) or an acceptor dye (Atto® 647N) in
order to study the intra-oligomer resonance energy transfer (FRET) down at the single
molecule level. The interaction of donor- and acceptor-labeled monomers to form
oligomeric aggregates is therefore detected by the presence of FRET. We employed a
multiparameter, dual-color excitation SMF approach to extract the size distributions and
the intra-oligomer FRET efficiency of the detected oligomers.
Using this technique, we detected the striking presence of aggregated species under
aggregation conditions, but yet in the absence of incubation. These species were small in
size, mostly dimers. This initial hypothesis was later supported by cross-linking
experiments.
Likewise, the low efficiency of the intra-oligomer FRET suggested a loose molecular
organization of the oligomers. Even at the low concentration ranges employed in the SMF
experiments, the formation of these labile, small, low-FRET oligomers was clearly
detected. A simple model was used to determine the value of the dissociation equilibrium
constant of these oligomeric species via the rate of the single oligomer events at different
protein concentrations.
Further research is in progress to resolve the role and importance of these genuine, early
oligomers within the mechanism of amyloid fibril formation.
P95
CHARACTERIZATION OF THE C2 DOMAINS OF
RABPHILIN 3A AND SYNAPTOTAGMIN 1
Dolores Pérez-Sánchez, Teresa Coronado-Parra, Jaime Guillén, Juan C. Gómez-Fernández,
Nuria Verdaguer and Senena Corbalán-García
Departamento de Bioquímica, Facultad de Veterinaria, Universidad de Murcia .
Proteins containing C2 domains are the sensors for Ca+2 in myriad of secretory pathways.
Here, we have determined the structure of this domain in complex with PI(4,5)P2 and IP3
at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed
by strands β3-β4 is involved in the interaction with the phosphoinositides. A comparative
study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3,
whereas the C2B domain cannot discriminate among any of the diphosphorylated forms.
Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1
indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin
1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural
explanation for the ability of different C2 domains to pull plasma and vesicle membranes
close together in a Ca+2-dependent manner and reveal how this family of proteins can use
subtle structural changes to modulate their sensitivity and specificity to various cellular
signals.
P96
STRUCTURAL CHARACTERIZATION OF TOMATO ABA RECEPTORS.
A FRAMEWORK TO DESIGN NEW AGONISTS AND ANTAGONISTS
TO FIGHT AGAINST ABIOTIC STRESS
Moreno Alvero, M., Yunta Yanes, C., González Rubio, J.M.
Martínez Ripoll, M., Albert, A.
INSTITUTO DE QUÍMICA FÍSICA “ROCASOLANO”. CENTRO SUPERIOR DE
INVESTIGACIONS CIENTÍFICAS (CSIC)
mmoreno@iqfr.csic.es
Plants have to endure adverse environmental conditions due to their sessile nature, for
that reason evolution has provided specialized protein networks to resist abiotic stress.
The research on the molecular mechanism underlying these networks is important since
it provides opportunities to generate or manage crops with enhanced resistance for our
benefit.
The plant stress hormone abscisic acid (ABA) regulates many of the stress adaptive plant
processes. The coordinated action of the plant PYR (pyrabactin resistance) family of
protein abscisic acid receptors, their interacting protein phosphatase type 2C substrates
(PP2C) and a group of protein kinases SnRK2 function together in decoding ABA signals
elicited by different environmental stimuli. Biochemical, cellular and structural studies
have shown that the PP2C-PYR interaction controls the phosphorylation state of SnRK2
and is fundamental for the regulation of the system. Hence, a balance between the
activities of negative (PP2C) and positive (SnRK2) regulators fine-tunes the cell response
in an ABA-dependent manner. The knowledge of the different genomes of crops plants
allows the identification and characterization of ABA receptors and phosphatases in
plants with commercial value. With this aim we have identified and characterized the
family of 14 ABA receptors in tomato.
The generation of agonist and antagonist molecules of ABA has been revealed as a very
effective tool to control plants response in stress situations. To gain knowledge in this
subject, particularly, on the chemical approach for crop enhancement, we have solved the
crystal structure of the unbounded tomato receptor sl08 and its complexes with ABA and
the agonist Quinabactin. Our data provide a totally new framework for the design of
antagonist molecules using ABA binding pocket.
Our work shows the potential applications of this knowledge to enhance plants abiotic
stress resistance.
P97
iMODS: FAST EXPLORATION OF MACROMOLECULAR
COLLECTIVE MOTIONS
José Ramón Lopéz-Blanco, Erney Ramírez and Pablo Chacón
Department of Biological Chemical Physics, Rocasolano Physical Chemistry Institute, CSIC,
Serrano 119, Madrid 28006, Spain.
iMODS is a web-based tool to approximate protein and nucleic acid flexibility using
normal mode analysis in internal coordinates [1]. Given an input atomic structure, the
server provides a fast and powerful tool to model, visualize and analyze functional
collective motions. Vibrational analysis, motion animations and morphing trajectories
can be easily carried out at different scales of resolution. The server is very versatile, nonspecialists can rapidly characterize potential conformational changes whereas advanced
users can select between multiple coarse-grained representations and elastic network
potentials. It includes advanced visualization capabilities for illustrating molecular
flexibility based on affine-models and vector field representations. The visualization
engine is also compatible with HTML5 and WebGL capabilities ensuring full
accessibility to all devices. The web server can be freely accessed at
http://imods.chaconlab.org.
López-Blanco JR, Garzón JI, Chacón P. (2011) iMod: multipurpose normal mode
1.
analysis in internal coordinates. Bioinformatics. 27 (20): 2843-2850.
P98
PLANT CYTOCHROME c1 EXHIBITS TWO BINDING SITES
FOR CYTOCHROME c WITH DISTINCT AFFINITIES
Blas Moreno-Beltrán1*, Irene Díaz-Moreno1, Katiuska González-Arzola1,
Adrián Velázquez-Campoy2, Miguel A. De la Rosa1 and Antonio Díaz-Quintana1
Instituto de Bioquímica Vegetal y Fotosíntesis, cicCartuja, Universidad de Sevilla - CSIC,
Avda. Américo Vespucio 49, Sevilla 41092, Spain
qzaida@us.es
2
Institute of Biocomputation and Physics of Complex Systems (BIFI) - Joint Unit BIFIIQFR (CSIC),
Universidad de Zaragoza, Mariano Esquillor s/n, 50018 Zaragoza, Spain; Departamento de Bioquímica y
Biología Molecular y Celular, Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain;
Fundación ARAID, Government of Aragon, María de Luna 11, 50018 Zaragoza, Spain
1
In plants, cytochrome c participates is channeled between membrane complexes III and
IV to carry electrons between from the first one to the second [1]. However, the mode
cytochrome c behaves as regards the dynamics of the respiratory complexes remains
unclear. Here, we report NMR-driven docking computations showing two well-defined
binding sites for cytochrome c at the head soluble domain of cytochrome c1, namely a
non-productive (or distal) site with a long heme-to-heme distance, and a functional (or
proximal) site with the two heme groups close enough as to allow electron transfer.
These binding sites exhibit clearly different equilibrium dissociation constants for the
reduced species, still under the micromolar range, so revealing the transient nature of
such a respiratory complex. Although the docking of cytochrome c at the distal site
occurs at the proximity to the interface between cytochrome c1 and the Rieske subunit,
it is fully compatible onto the complex III structure. In our model, the extra distal site in
complex III locates near complex IV according to the plant respirasome structure. This
may facilitate shuttling of electrons towards the oxidase while averting cytochrome c
molecules drift into the bulk mitochondrial intermembrane space, thereby enhancing the
efficiency of the electron transport chain.
Acknowledgement: Financial support was provided by the Spanish Ministry of Economy and
Competiveness (Grant No. BFU2009-07190/BMC and BFU2012-31670/BMC) and by the
Andalusian Government (Grant PAI, BIO198). BMB was awarded with a PhD fellowship (AP20094092), co-funded by European Social Fund-ERDF (2007-2013). We thank Bio-NMR Research
Infrastructure (Project BIO-NMR-00130), co-funded under the 7th Framework Programme of the
EC (FP7/2007–2013). We also thank CITIUS NMR Facility at University of Seville.
Reference
[1] Genova, M.L. and Lenaz, G. (2013) A critical appraisal of the role of respiratory supercomplexes in
mitochondria. Biol Chem, 394, 631-639.
LECTURES BY PRIZE AWARDEES
Annual Awards Ceremony
Chairperson: Juan C. Gómez Fernández (UM, Murcia)

Bruker Prize:
Visualizing the 3D structure of transient macromolecular interactions using EM.
Óscar Llorca, (CIB-CSIC, Madrid)

SBE-40/Pérez-Payá Prize:
Recognition and resection of double-stranded DNA breaks: a single-molecule approach.
Fernando Moreno-Herrero (CNB-CSIC, Madrid)

SBE-33/Elsevier Prize:
Mechanobiochemistry of the giant protein titin.
Jorge Alegre-Cebollada (Columbia University, NY)
VISUALIZING THE 3D STRUCTURE OF TRANSIENT
MACROMOLECULAR INTERACTIONS USING ELECTRON MICROCOPY
Oscar Llorca
Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC),
Madrid, Spain
ollorca@cib.csic.es
The structural analysis of short-lived macromolecular interactions by three-dimensional
electron microscopy (3D-EM) is challenged by the tendency of these complexes to fall
apart. These can also display flexible/transient conformations, which, if undetected and/or
adequately handled, can result in solving “incorrect” structures. I will describe our
experience undertaking the structural analysis of transient interactions and conformations,
including cross-linking, use of mutations and classification of conformational transitions
co-existing in the same sample by image processing.
I will describe examples on two on-going projects, nonsense-mediated mRNA decay
(NMD) (1) and the complement system (2, 3). NMD is a post-transcriptional surveillance
mechanism that recognizes and degrades mRNAs containing premature translation
termination codons (PTCs). NMD is initiated by the crosstalk between a ribosome, the
exon-junction-complex (EJC) and a collection of protein factors, forming transient
interactions and complexes that we are trying to describe and understand.
Complement is a major component of innate immunity with crucial roles in microbial
killing. We seek to provide a full understanding of the structural mechanisms that underlie
complement regulation. Complement operates throughout the formation of unstable,
short-lived, macromolecular complexes that result from the interaction between proteins
undergoing proteolysis and large conformational changes. This project is performed in a
close collaboration with Santiago Rodriguez de Córdoba (CIB), as part of a consortium
funded by the Autonomous Government of Madrid.
Acknowledgements: This work was supported by the Autonomous Region of Madrid (S2010/BMD2316), the “Ramón Areces” Foundation, the Spanish Government (SAF2011-22988), a Juan de la
Cierva contract (to R. Melero) and a Sara Borrell contract (CD09/00282, to M. Alcorlo).
References
[1] Melero R, Buchwald G, Castaño R, Raabe M, Gil D, Lázaro M, Urlaub H, Conti E, Llorca O, “The
cryo-EM structure of the UPF-EJC complex shows UPF1 poised toward the RNA 3' end”, Nat Struct
Mol Biol. 19(5):498-505, 2012.
[2] Alcorlo M, Tortajada A, Rodríguez de Córdoba S, Llorca O, “Structural basis for the stabilization of
the complement alternative pathway C3 convertase by properdin”, Proc Natl Acad Sci U S A.
13;110(33):13504-9, 2013.
[3] Alcorlo M, Martínez-Barricarte R, Fernández FJ, Rodríguez-Gallego C, Round A, Vega MC, Harris
CL, de Cordoba SR, Llorca O, “Unique structure of iC3b resolved at a resolution of 24 Å by 3Delectron microscopy”, Proc Natl Acad Sci U S A. 108(32):13236-40, 2011.
RECOGNITION AND RESECTION OF DOUBLE-STRANDED DNA BREAKS:
A SINGLE-MOLECULE APPROACH
Fernando Moreno-Herrero
Department of Macromolecular Structures, Centro Nacional de Biotecnología
(CNB-CSIC), Darwin 3, 28049, Madrid, Spain.
fernando.moreno@cnb.csic.es
Proper development of life relies on the ability of cells to repair the many double-stranded
DNA breaks (DSBs) that occur during normal metabolism. Improper DSB repair may
result in loss of chromosome structural integrity and genomic instability, and is associated
with developmental defects, deficiencies of the immune system and cancer
predisposition. Cells possess robust mechanisms to repair DNA breaks. One such DNA
repair mechanism is homologous recombination where the sister chromatid is used as a
template for faithful repair. Recombinational repair is initiated by the recognition and
resection of a duplex DNA end to form a 3´-terminated single-stranded DNA overhang.
This task is carried out by different proteins acting as truly nano-machines cutting,
pasting, or copying fragments of DNA in a highly efficient manner.
In this talk, I will describe our recent discoveries in DNA repair that have provided an
exquisite understanding of DNA-end resection by helicases and nucleases. In particular,
I will focus on the mechanism of DNA resection and hotspots sequence (Chi) recognition
of the model system AddAB from Bacillus subtilis. We employed Atomic Force
Microscopy and Magnetic Tweezers (MT) to image the products of the resection reaction
and to monitor the real-time activity of the AddAB molecular motor [1] [2] [3]. We found
that AddAB couples double-stranded DNA translocation and unwinding using either the
SSB protein as a cofactor or through the interaction with Chi sequences and the formation
of a single-stranded DNA loop. Additionally, our MT experiments revealed that AddAB
pauses at Chi sequences and resumes translocation on average at a slower pace. Our
findings set the basis for a comprehensive model where pausing of the helicase at Chi
acts as a selectivity filter for bona fide Chi recognition.
Acknowledgements: This work has been sponsored by a Starting Grant from the European Research
Council ref 206117, and by a grant from the Spanish Ministry of Economy and Competitiveness ref
FIS2011-24638.
References
[1] J.T. Yeeles, K. van Aelst, M.S. Dillingham, and F. Moreno-Herrero, Recombination hotspots and
single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB
helicase-nuclease. Molecular Cell, 42, 806-816 (2011).
[2] C. Carrasco, N.S. Gilhooly, M.S. Dillingham, and F. Moreno-Herrero, On the mechanism of
recombination hotspot scanning during double-stranded DNA break resection. Proc Natl Acad Sci U
S A, 110, E2562-71 (2013).
[3] C. Carrasco, M.S. Dillingham, and F. Moreno-Herrero, Single molecule approaches to monitor the
recognition and resection of double-stranded DNA breaks during homologous recombination. DNA
Repair (Amst) (2014) On-line doi: 10.1016/j.dnarep.2014.02.002.
MECHANOBIOCHEMISTRY OF THE GIANT PROTEIN TITIN
Jorge Alegre-Cebollada
Department of Biological Sciences. Columbia University, New York, NY 10027, USA.
Department of Vascular Biology and Inflammation, CNIC, 28029 Madrid, Spain
jalegre@cnic.es
Mechanical forces are the number one protein denaturant in cells. How proteins unfold
and refold under force has strong implications in physiological responses such as muscle
elasticity.
I will present the latest developments in single-molecule force spectroscopy by Atomic
Force Microscopy and Magnetic Tweezers, the techniques of choice to examine
mechanical protein folding and unfolding.
I will discuss how the elasticity of muscle is modulated biochemically through
modification of cryptic residues that become exposed upon unfolding of titin Ig domains.
I will outline anticipated future developments in the new field of mechanobiochemistry.
PLENARY LECTURES
PL1 (Sponsored by Consolider-Ingenio)
TRP channels: what are they and why are they important for understanding
polymodality of ion channels?
Baruch Minke
PL2 (European Biophysical Societies Association Lecture)
Engineering tunnels and gates in enzymes.
Jiri Damborsky
PL3 (Sponsored by Elsevier)
Membrane biophysics intersecting biological function.
Richard Epand
PL4
Unraveling the mechanism of molecular motors by using micromanipulation methods.
María Mañosas
PL5
Is it possible to fully understand a living system in a quantitative manner?
Luis Serrano
TRP CHANNELS: WHAT ARE THEY AND WHY ARE THEY IMPORTANT
FOR UNDERSTANDING POLYMODALITY OF ION CHANNELS?
Baruch Minke and Maximilian Peters
Departments of Medical Neurobiology, the Institute of Medical Research Israel-Canada (IMRIC),
The Edmond and Lily Safra Center for Brain Sciences (ELSC)
Faculty of Medicine of the Hebrew University, Jerusalem 91120, Israel.
Transient receptor potential (TRP) channels constitute a large superfamily of polymodal
channel proteins with diverse roles in many transduction and sensory pathways. The TRP
superfamily, which is conserved through evolution, consists of seven subfamilies and its
members are expressed in many cell types. These channels participate in most sensory
modalities and they either open directly in response to ligands or physical stimuli or, indirectly,
downstream of a signal transduction cascade. Currently, the gating mechanism of TRP
channels is unclear.
We explored the role of critical amino acids in the highly conserved pore region of the
Drosophila TRPC channels, TRP and TRPL. We generated specific point mutations, which affect
the activation state of the channel and may explain how the channel-lipid interactions
determine the activation state of the channel. The alignment of transmembrane region 5 of
TRP channels reveals highly conserved amino acids.
To get insights into the gating mechanism and validate homology models, we bioinformatically
analyzed TRPL and identified the area in transmembrane region 5 as a critical regulator of TRPL
activity. By a series of mutations we could generate TRPL mutants which are constitutively
active in HEK293 cells where TRPL is normally not active, or show different responses to the
non-specific TRP modulator polyunsaturated fatty acid. By mutating amino acid pairs we were
able to gain insights in the orientation of the transmembrane domains and validate homology
models.
We conclude that despite sequence differences and evolutionary separation, the gating
mechanism and overall structure of the TRPC channel, TRPL, and the widely investigated
polymodal TRPV1 channel is conserved.
ENGINEERING TUNNELS AND GATES IN ENZYMES
Jiri Damborsky, Jan Brezovsky, Zbynek Prokop, Radka Chaloupkova, Sergio Manuel Marques,
Shubhangi Kaushik, Jaroslav Bendl, Petra Szelcsanyiova, Jan Stourac
Loschmidt Laboratories, Department of Experimental Biology and the Research Centre for
Toxic Compounds in the Environment, Masaryk University, Brno, Czech Republic
jiri@chemi.muni.cz
Protein structures contain a complex system of voids, making up specific features - clefts,
pockets, cavities, channels and tunnels. These features are essential for the migration of
solvents, ions and small molecules through the protein structure and represent the natural hot
spots for protein engineering. This migration is often controlled by highly dynamical structures
called molecular gates. In this lecture, we will present: (i) examples of protein families
possessing tunnels1 and gates2, (ii) software tools3 available for detection and analysis of
tunnels and gates, (iii) success stories from engineering tunnels for catalytic activity4,
enantioselectivity5 and stability6. We will demonstrate applicability of the software tools
HOTSPOT WIZARD7 and CAVER8 for analysis and design of dynamical access pathways and will
advocate the design of tunnels and gates as a powerful strategy for construction of novel
biocatalysts.
References
[1] Prokop, Z., et al., Engineering of Protein Tunnels. In. Lutz, S., Bornscheuer, U.T. (Eds.),
Protein Engineering Handbook, Wiley-VCH, Weinheim, 421-464, 2012.
[2] Gora, A., et al., Gates of Enzymes, Chemical Reviews, 113, 5871-5923, 2013.
[3] Brezovsky, J., et al., Software Tools for Identification, Visualization and Analysis of Protein
Tunnels and Channels, Biotechnology Advances, 31, 38-49, 2012.
[4] Pavlova, M., et al., Redesigning Dehalogenase Access Tunnels as a Strategy for Degrading
an Anthropogenic Substrate, Nature Chemical Biology, 5, 727-733, 2009.
[5] Prokop, Z., et al., Enantioselectivity of Haloalkane Dehalogenases and its Modulation by
Surface Loop Engineering, Angewandte Chemie, 49, 6111-6115, 2010.
[6] Koudelakova, T., et al., Engineering Protein Resistance to Organic Co-solvent and Elevated
Temperature by Access Tunnel Modification, Angewandte Chemie International Edition, 52,
1959-1963, 2013.
[7] Pavelka, A., et al., HotSpot Wizard: a Web Server for Identification of Hot Spots in Protein
Engineering, Nucleic Acids Research, 37, W376-W383, 2009.
[8] Chovancova, E., et al., CAVER 3.0: A Tool for Analysis of Transport Pathways in Dynamic
Protein Structures, PloS Computational Biology, 8, e1002708, 2012.
MEMBRANE BIOPHYSICS INTERSECTING BIOLOGICAL FUNCTION
Richard M. Epand
McMaster University, Hamilton, Ontario, Canada
epand@mcmaster.ca
Membrane biophysics encompasses studies both of the bulk material properties of the
membrane as well as studies of the properties and functions of individual molecular components
of membranes. The relationship of membrane proteins that include receptors, membranebound enzymes and transporters to biological function is generally obvious. The activity of these
proteins is often modulated by the bulk properties of the membrane, so the two aspects of bulk
properties and molecular properties, are not completely independent of each other but are
inter-related. Among the bulk properties of the membrane that have been shown to be
important for biological function include membrane curvature and membrane domain
formation. Membrane curvature properties include both the physical curvature of the
membrane as well as the intrinsic curvature. The differences between the two will be discussed,
as well as how they can modulate protein function. In addition to the mean curvature, there is
also Gaussian curvature that is thought to have an important role in membrane fusion. The other
physical property we will discuss is membrane domain formation. It is generally agreed that
biological membranes are not homogeneous, but there are regions in which the concentration
of certain components is increased. These are referred to as domains, but the nature and
composition of these domains is currently being refined. The particular type of domain termed
a “raft” has attracted particular attention as a possible platform in which signal transduction
proteins can effectively interact with one another. Bilayer lipid asymmetry is another aspect of
the inhomogeneity of a membrane that is associated with membrane function. Finally, there is
the rapidly evolving field of membrane protein structure that has given many experimental
challenges. A wide variety of methods have been applied to this problem including NMR, X-ray
diffraction and electron microscopy imaging. With the development of new detector methods,
there is now a promise of rapid advances in generating structures of membrane proteins without
the need for use of detergents or crystallization.
Acknowledgements: This work has been sponsored by the Canadian Natural Sciences and
Engineering Research Council, grant 9848.
UNRAVELING THE MECHANISM OF MOLECULAR MOTORS
BY USING MICRO MANIPULATION METHODS
M. Mañosas1, MM Spiering2, SK Perumal2, P. Bianco3, F. Ding4, F. Ritort1,
JF Allemand4, D Bensimon4, SJ Benkovic2, V. Croquette4
1
Departament de Física Fonamental, Facultat de Física,
Universitat de Barcelona, Barcelona 08028, Spain,
mmanosas@ub.edu
2
Department of Chemistry, The Pennsylvania State University, University Park,
16802 Pennsylvania, USA
3
Department of Microbiology and Immunology, Center for Single Molecule Biophysics,
University at Buffalo, Buffalo, 14214, New York USA
4
Laboratoire de Physique Statistique, Ecole Normale Supérieure, UPMC Univ. Paris 06,
Université Paris Diderot, CNRS, 24 rue Lhomond, 75005 Paris, France
Single-molecule micromanipulation methods have shed new light on DNA protein
interactions. In particular these methods have provided novel insights on the mechanisms
of molecular motors that convert chemical energy (e.g. the energy released in the
hydrolysis of ATP) into mechanical work. In this talk I will describe the use of magnetic
traps for the investigation of DNA processing motors involved in DNA replication and DNA
repair. In these assays magnetic traps are used to mechanically manipulate a DNA
molecule and follow in real time the activity of different DNA molecular motors. The
applied mechanical force allows either to assist or to hinder motor activities revealing the
mechanisms of individual motors [1-3] as well as their coordinated action when
processing DNA, such as during DNA replication and repair [4-6].
Acknowledgements: This work has been sponsored by Human Frontiers Science Program, European
Research Council and Ministerio de Economia y Competividad.
References
[1] M Manosas, XG Xi, D Bensimon, V Croquette. Active and passive mechanisms of helicases. Nucl. Acids
Res. 38(16): 5518 - 552, 2010.
[2] M Manosas, MM Spiering, F Ding, D Bensimon, JF Allemand, SJ Benkovic, V Croquette. Mechanism of
strand displacement synthesis by DNA replicative polymerases. Nucl. Acids Res. 40(13): 6174-6186,
2012.
[3] M Manosas, SK Perumal, P Bianco, F Ritort, SJ Benkovic, V Croquette. RecG and UvsW catalyse robust
DNA rewinding critical for stalled DNA replication fork rescue. Nat. Comm. 4 2368: 1-11, 2013.
[4] M Manosas, MM Spiering, Z Zhuang, SJ Bencovik, V Croquette. Coupling DNA unwinding with primer
synthesis in the bacteriophage T4 primosome. Nat. Chem. Bio. 5: 904 - 912, 2009.
[5] M Manosas, MM Spiering, F Ding, V Croquette, SJ Benkovic. Collaborative coupling between
polymerase and helicase for leading-strand synthesis. Nucl. Acids Res. 40(13): 6187-6198, 2012.
[6] M Manosas, SK Perumal, V Croquette, SJ Benkovic. Direct observation of stalled fork restart via fork
regression in the T4 replication system. Science 338(6111): 1217-1220, 2012.
IS IT POSSIBLE TO FULLY UNDERSTAND A LIVING SYSTEM
IN A QUANTITATIVE MANNER?
Luis Serrano
CRG-Centre for Genomic Regulation, Edif. PRBB, c/ Dr. Aiguader, 88
08003 Barcelona, Spain. Tel. +34 93 316 01 98; Fax +34 93 316 00 99
By combining all possible –omics approaches with Bioinformatics analysis and computer
modeling we are trying to see if it is possible to understand in a quantitative manner a
living system.
In parallel, and with this knowledge and in collaboration with Sanofi, we are engineering
Mycoplasma pneumoniae for therapeutic applications.
INVITED LECTURES
S1: Supramolecular assemblies.
Chairperson: José López Carrascosa (CNB, CSIC, Madrid)
IL1.1. Mechanisms of microtubule plus-end tracking proteins and centriolar proteins.
Michel Steinmetz (Paul Scherrer Institute, Villigen PSI, Switzerland)
IL1.2. The protein folding pathway: a coordinated network of molecular machines.
José M. Valpuesta (CNB, CSIC, Madrid)
IL1.3. Capturing working conformations for a large multifunctional enzyme.
Mikel Valle (CIC BioGUNE, Bilbao)
IL1.4. Targeting protein assembly machines of the tubulin-FtsZ superfamily with small
molecules.
José M. Andreu (CIB, CSIC, Madrid)
STRUCTURAL BASIS OF THE 9-FOLD SYMMETRY OF CENTRIOLES
Michel O. Steinmetz
Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen PSI, Switzerland,
michel.steinmetz@psi.ch
The centriole and the related basal body is an ancient organelle characterized by a
universal 9-fold radial symmetry and which is critical for generating cilia, flagella and
centrosomes. The mechanisms directing centriole formation are not understood and
represent a fundamental open question in biology. We recently demonstrated that the
centriolar protein SAS-6 forms rod-shaped homodimers that interact through their Nterminal domains to form oligomers. We established that such oligomerization is essential
for centriole formation in eukaryotic cells. We further generated a structural model of
SAS-6, in which nine homodimers assemble into a ring from which nine coiled-coil rod
domains radiate outwards. Moreover, we demonstrated that recombinant SAS-6 selfassembles into structures akin to the central hub of the cartwheel, which serves as a
scaffold for centriole formation. Overall, our findings established a structural basis for
the universal nine-fold symmetry of centrioles [1].
Acknowledgements: This work has been sponsored by grants from the Swiss National Science
Foundation (Sinergia CRSII3_125463), Oncosuisse (OCS KLS 02024-02-2007) and ERC (AdG
233335), and by fellowships from JSPS, EMBO (ALTF-667-2007), Wellcome Trust and Marie Curie.
Reference
[1] Kitagawa, D., Vakonakis, I., Olieric, N., Hilbert, M., Keller, D., Olieric, V., Bortfeld, M., Erat, M.C.,
Flückiger, I., Gönczy, P., Steinmetz, M.O. Structural basis of the nine-fold symmetry of centrioles.
Cell, 144, 1-12, 2011.
THE PROTEIN FOLDING PATHWAY:
A COORDINATED NETWORK OF MOLECULAR MACHINES
José María Valpuesta
Centro Nacional de Biotecnología (CNB-CSIC). Darwin, 3. 28049 Madrid. Spain
jmv@cnb.csic.es
Molecular chaperones are a group of proteins devoted to either assisting the folding
of other proteins or to their degradation. The former they generally do it by protecting
the aggregation-prone regions of these proteins, thus allowing them to reach their
native conformation using the information encoded in their own amino acid
sequences. As for the later, this is achieved by the interaction of the
chaperone:substrate complex with certain factors (cochaperones) which direct the
complex to the proteasome degradation pathway. In both processes, chaperones can
work by themselves but in most cases their function is performed by the coordinated
concourse of different chaperones, which form transient complexes, thus acting like
“assembly lines” that make more efficient the protein folding and degradation
processes.
The talk will be devoted to analyze some of these “assembly lines”, a work that has
been mainly carried out by electron microscopy, a technique suited for the study of
transient protein complexes generated during the folding and degradation processes.
CAPTURING WORKING CONFORMATIONS FOR
A LARGE MULTIFUNCTIONAL ENZYME
Mikel Valle1, David Gil1, Melisa Làzaro1, Linda PC Yu2, Liang Tong2 and Gorka Lasso1
1
CIC bioGUNE, Derio, Spain.
email: mvalle@cicbiogune.es
2
Columbia University, New York, USA.
Pyruvate carboxylase (PC) is a biotin-containing multifunctional enzyme that
carboxylates pyruvate into oxaloacetate in two sequential chemical reactions. PC is a
130kDa multifunctional single-chain enzyme in eukaryotes and most bacteria and is
active only as a homotetramer. The biotin carboxylase (BC) and the carboxyltransferase
(CT) domains contain the two active sites necessary for the enzymatic activity while the
biotin carboxyl carrier (BCCP) domain couples both active sites. This domain contains a
covalently bound biotinyl group which is carboxylated in the BC domain in a reaction
that requires ATP hydrolysis. Subsequently, the BCCP domain moves to the CT domain
where it transfers the carboxyl group to pyruvate, releasing oxaloacetate. During the
enzymatic activity the BCCP domain travels from BC to CT active sites, which are at a
distance of around 75 Å [1,2]. Previous cryoEM work with PC has unveiled some of the
mechanisms of allosteric control [3], but a clear picture of the transitions between BC and
CT reactions is still missing. Now, by combining cryoEM and sorting techniques, we
have explored the conformational variations of PC while the enzyme performs the full
chemical reactions, i.e., in the presence of all the substrates and cofactors and with no
synchronization. We detect previously defined symmetric and asymmetric architectures,
demonstrating that PC maps both arrangements by large conformational changes.
Furthermore, we observe that each configuration is coupled to one of the two consecutive
enzymatic reactions. The findings describe the structural transitions relevant for the
allosteric control of the multifunctional PC, and demonstrate that by cryoEM and
classification we can characterize freely working macromolecules.
Acknowledgements: This work has been sponsored by grants BFU2012-34873 (to MV) and DK067238
( to LT).
References:
[1] St Maurice, M., Reinhardt, L., Surinya, K.H., Attwood, P.V., Wallace, J.C., Cleland, W.W., and
Rayment, I. Science, 317 (2007) 1076-1079.
[2] Xiang, S., and Tong, L. Nat. Struct. Mol. Biol., 15 (2008), 295-302.
[3] Lasso, G., Yu, L., Gil, D., Xiang, S., Tong, L., Valle, M. Structure, 18 (2010) 1300-1310.
TARGETING PROTEIN ASSEMBLY MACHINES OF THE
TUBULIN-FtsZ SUPERFAMILY WITH SMALL MOLECULES
José M. Andreu
Centro de Investigaciones Biológicas-CSIC, Madrid, Spain
j.m.andreu@cib.csic.es
Tubulin-like proteins typically associate head to tail into similar polar protofilaments
with a 4 nm axial spacing between the structurally homologous monomers.
Protofilaments form different types of dynamic polymers and subcellular structures,
including the mitotic spindle or the bacterial division ring. Polymer assembly and
disassembly is thus directly related to their function, serving as tracks, scaffolds, or even
producing motility without the assistance of motor proteins. Assembly involves the
formation of a subunit-subunit interface where the GTP nucleotide gets buried and is
hydrolyzed, which then triggers disassembly, followed by subunit reloading with GTP.
In addition, tubulin and FtsZ have built-in structural switches between their free and
assembled states. I will briefly present a new structural mechanism of tubulin assembly
inhibition by powerful anticancer drugs (1, 2), then focus on antibacterial inhibitors of
FtsZ and its assembly switch.
FtsZ is the organizer for cell division in most bacteria, where it forms the contractile
Z-ring that recruits the other divisomal protein. FtsZ is a target for new antibiotics, needed
to fight the widespread emergence of resistant pathogens (3). FtsZ self-assembly can be
selectively inhibited with modified nucleotides (4). In a further step, we have replaced
FtsZ´s GTP with synthetic inhibitors of bacterial cell division (5). We tested compounds
from the literature, virtual screening and a synthetic library. Three small molecules bind
Bacillus subtilis FtsZ monomers with micromolar affinities, impair normal FtsZ
assembly, the localization of FtsZ-GFP to the Z-ring, and effectively inhibit growth of
antibiotic-resistant pathogens Staphylococcus aureus and Enterococcus faecalis.
Systematic chemical modification has defined their essential features for binding, leading
to new analogs with improved affinity-activity that selectively inhibit FtsZ versus tubulin.
The assembly switch of FtsZ has been proposed to involve the opening of the cleft
between its C-terminal and nucleotide binding domains, where the antibacterial agent
PC190723 binds and is thought to allosterically stabilize the FtsZ protofilament. Large
scale molecular dynamics simulations from representative FtsZ filament crystal structures
show a nucleotide-regulated hinge motion between consecutive FtsZ monomers, monitor
the FtsZ assembly switch opening and closing the cleft, and reproduce the filament
stabilization by PC190723 (6). A fluorescent analog of this ligand preferentially binds to
assembled FtsZ, which supports the cleft-opening mechanism for the FtsZ assembly
switch and provides a tool to screen for new antibacterials targeting the FtsZ inter-domain
region (7).
Acknowledgment: Supported by grants BFU2011-23416 and CMS2010/BMD-2353.
[1] Pera B. et al. and Andreu J.M. (2013) ACS Chem. Biol. 8, 2084-94.
[2] Prota A. et al. and Steinmetz M.O. (2014) submitted.
[3] Schaffner-Barbero C. et al. and Andreu J.M. (2012) ACS Chem. Biol. 7, 269-77.
[4] Marcelo F. et al. and Andreu J.M. (2013) JACS 135, 16418-28.
[5] Ruiz-Avila L.B. et al. and Andreu J.M. (2013) ACS Chem. Biol. 8, 2072-83.
[6] Ramirez-Aportela E. et. al. and Chacón P. (2014) submitted & Poster at this meeting.
[7] Huecas S. et al. and Andreu J.M., Abstract, this meeting.
INVITED LECTURES
S2 (Consolider-Ingenio): Receptors, channels and transporters.
Chairperson: Antonio V. Ferrer Montiel (IBMC-UMH, Elche, Alicante)
IL2.1. hERG cytoplasmic domains motifs that determine traffic and gating properties
of the channel.
Francisco Barros (Uniovi, Oviedo)
IL2.2. New insights into the role of the cyclin M family in magnesium homeostasis.
Alfonso Martínez de la Cruz (CIC BioGUNE, Bilbao)
IL2.3. The potassium channel KcsA : A model for developing useful tools in the
understanding of potassium channels and design new potential drugs.
Asia Fernández-Carvajal (IBMC-UMH, Elche, Alicante)
IL2.4. Aquaporin channels regulation: targeting new players in old diseases.
Graça Soveral (iMed.UL, Lisbon, Portugal)
hERG CYTOPLASMIC DOMAINS THAT DETERMINE GATING
AND TRAFFIC PROPERTIES OF THE CHANNEL
Francisco Barros, Angeles Machín, Jorge Fernández-Trillo, Pedro Domínguez and Pilar de la
Peña
Department of Biochemistry and Molecular Biology, University of Oviedo, 33006 Oviedo, Asturias, Spain
The distinctive gating properties of the voltage-dependent potassium channel
human ether-á-go-go-related gene (hERG, Kv11.1 or KCNH2) determine its contribution
to cardiac repolarization and are important in setting the electrical behavior of a variety
of cells. There are numerous reports indicating that such gating characteristics are
strongly influenced by cytoplasmic regions that in hERG account for around 80% of the
protein. However, details of the structural architecture of these domains and the precise
mechanism(s) for which their modulation of gating and trafficking properties takes place
are still lacking. Using site-directed mutagenesis and functional characterization of
deleted channels we find that the conserved eag N-terminal domain at the beginning of
the hERG N-terminus and the exclusive proximal domain following it up to the S1
transmembrane segment are determinants of the remarkably slow deactivation and
activation gating, respectively. We have also demonstrated that hERG deactivation
slowing of eag domain-truncated channels is restored by a recombinant eag/PAS
fragment, and that both the FRET between this fragment and the channel core and the
slowing of closing are similarly affected by structural alterations either in the N-terminal
hERG tail or in the intracellular S4-S5 linker, pointing to the existence of interaction(s)
between them. Indeed, using a site-directed cysteine mutagenesis and disulfide chemistry
approach we have demonstrated the existence of a physical proximity between the N-tail
and both the S4-S5 linker and the C-linker region of the channel carboxy terminus. The
state-dependence of the crosslink also indicated that this disulfide bonding may occur
during conformational rearrangements taking place following excursions between the
open/inactive and closed states of the protein. Finally, we have checked the ability of
different inserted hERG C-terminus segments to retain intracellularly a hormone receptor
predominantly located to the plasma membrane. This allowed us to identify a hERG Cterminal coiled-coil sequence responsible for limiting receptor delivery to the membrane.
Furthermore, mutagenesis of this region significantly enhanced hERG channel trafficking
to the cell surface. Thus, our data highlight the crucial contribution of the cytoplasmic
regions of hERG (and other voltage-dependent K+ channels) to gating, trafficking,
stabilization and other functional aspects of channel behavior.
Acknowledgements: This work has been sponsored by grant BFU2009-11262 of the Spanish MICINN,
the Consolider-Ingenio project SICI CDS2008-00005 and a 2013 year Subvention (SV-PA-13ECOEMP-59) from Principado de Asturias. No financial support is presently available.
NEW INSIGHTS INTO THE ROLE OF THE CYCLIN M FAMILY
IN MAGNESIUM HOMEOSTASIS
Alfonso Martínez de la Cruz
CIC BioGUNE, Bilbao, Spain
Recent studies suggest cyclin M2 (CNNM2) to be part of the long-sought basolateral
Mg2+ extruder at the renal distal convoluted tubule, or as its regulator. Here, we explore
structural features and ligand binding capacities of the Bateman module of CNNM2
(residues 429-584), a domain involved in Mg2+ handling by the bacterial Mg2+ transporter
MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the
structural impact of the pathogenic mutation T568I, located in this region. Our crystal
structures reveal that nucleotides bind at only one of the two cavities present in
CNNM2429-584. Mg2+ favours ATP binding by alleviating the otherwise negative charge
repulsion existing between acidic residues and the polyphosphate group of ATP. In the
crystals CNNM2429-584 forms parallel dimers, commonly referred to as CBS modules.
Interestingly, nucleotide binding triggers a conformational change in the CBS module
from a twisted towards a flat disk-like structure that mostly affects the structural elements
connecting the Bateman module with the transmembrane region. We furthermore show
that the T568I mutation, which causes dominant hypomagnesemia, mimics the structural
effect induced by nucleotide binding. Our data suggest that T568I mutation exerts its
pathogenic effect in humans by constraining the conformational equilibrium of the CBS
module of CNNM2, which becomes “locked” in its flat form.
THE POTASSIUM CHANNEL KCSA: A MODEL FOR DEVELOPING
USEFUL TOOLS IN THE UNDERSTANDING OF POTASSIUM CHANNELS
AND DESIGN NEW POTENTIAL DRUGS
A. M. Fernández, M. L. Renart, E. Montoya, M. Giudici, M. L. Molina,
J. A. Poveda, J. A. Encinar, A. V. Ferrer-Montiel, and J. M. González Ros
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante, Spain
asia.fernandez@umh.es
Potassium channels are widely distributed in all kinds of living organisms and play critical roles
in a variety of physiological and pathophysiological processes. Our tenet is that by further
understanding K+ channels structure and function we can learn new concepts on the biology of
these important membrane proteins, and also potentiate their therapeutic use. We have used KcsA
as a model of potassium channel because this prokaryotic channel is structurally simpler than its
eukaryotic counterparts and it and has provided a structural-framework to understand selectivity,
ion-permeation, gating and pore-blocking. We have evaluated the contribution of pore cation
binding to ion permeation and selectivity features of KcsA and we have discovered that binding
of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation
properties. Ion binding stabilizes greatly the channel and, depending upon ion type and
concentration, leads to different conformations and ion binding affinities. High-affinity channel
states guarantee binding of specific ions and mediate ion selectivity but are nonconductive.
Conversely, low-affinity states would not discriminate well among different ions but allow
permeation to occur [1].
Another characteristic of many ion channels is their ability to assemble into supramolecular
complexes or clusters in which their function becomes modulated through homomeric or
heteromeric molecular interactions. Despite the apparent simplicity of KcsA channel, it has been
reported that the membrane-bound KcsA tetramer further assembles into still insufficiently
defined clusters, both in vitro [2] and in vivo [3]. Such clusters seem responsible for the adoption
of different modes of coupled gating, which causes large changes in channel function [2]. Indeed,
KcsA clustering is likely behind the complex activity patterns detected in many
electrophysiological studies. Again, KcsA might be an excellent model to identify structural
motifs responsible for clustering-mediating, channel–channel interactions or to establish the
mechanisms by which such interactions lead to coupled gating of ion channels.
We have also studied the process of rapid inactivation in K+ channels; which plays an important
role in shaping the electrical signaling properties of nerve and muscle cells.
The property of inactivating peptide of the Shaker B K + channel to block KcsA channel should
allow us to define a “pharmacophore” for a brand-new strategy of “structure-based” design of ion
channel ligands [4].
Acknowledgements: This work has been sponsored by grants from the Spanish MICINN BFU20080062/BMC, BFU2009-08346, BFU2011-25920 and Consolider-Ingenio 2010 CSD2-2008-00005.
References:
[1] ML Renart, E Montoya, AM Fernández, ML Molina, JA Poveda, JA Encinar, AV Ferrer-Montiel, J Gómez and
JM González Ros. Contribution of ion binding affinity to ion selectivity and permeation in KcsA, a model
potassium channel. Biochem. 8;51(18):3891-900, 2012.
[2] ML Molina, FN Barrera, AM Fernandez, JA Poveda, ML Renart, J.A Encinar, G Riquelme, JM Gonzalez-Ros,
Clustering and coupled gating modulate the activity in KcsA, a potassium channel model, J. Biol. Chem. 18837–
18848, 2006.
[3] J. Hegermann, J Overbeck, H Schrempf, In vivo monitoring of the potassium channel KcsA in Streptomyces
lividans hyphae using immuno-electron microscopy and energy filtering, Microbiology, 152, 2831–2841, 2006.
[4] ML Molina , Barrera FN, Encinar JA, Renart ML, Fernandez AM, Poveda JA, Santoro J, Bruix M, Gavilanes F,
Fernández-Ballester GF, Neira JL, González-Ros JM. (2008). N-type inactivation of the potassium channel kcsa
by the shaker b “ball” peptide: mapping the inactivating peptide binding epitope. J. Biol. Chem 283(26):1807685.
AQUAPORIN CHANNEL REGULATION:
TARGETING NEW PLAYERS IN OLD DISEASES
Graça Soveral1 and Angela Casini2
1
Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia,
Universidade de Lisboa, Lisboa, Portugal
gsoveral@ff.ul.pt
2
Dept. of Pharmacokinetics, Toxicology and Targeting, Research Institute of Pharmacy,
University of Groningen, The Netherlands
Aquaporins (AQPs) are membrane water channels involved in the transport of water
(strict aquaporins) and small solutes such as glycerol (aquaglyceroporins) involved in
many biological functions, including transepithelial fluid transport, cell migration and
proliferation, brain oedema and neuroexcitation, adipocyte metabolism and epidermal
water retention. In the last decade, the aquaporin field became a very hot area of research
with increasing physiological and medical implications. Human diseases caused by
aquaporin dysfunction unveiled these proteins as potential drug targets and opened new
perspectives to untangle mechanisms of disease [1].
We recently reported on the potent and specific inhibition of aquaglyceroporins by goldcontaining compounds [2]. Human AQP3 was strongly inhibited by gold(III) complexes,
with Auphen being the most potent in the series [3]. Computational modeling and site
directed mutagenesis revealed that Auphen blocks the channel by steric occlusion due to
interaction with Cys40 present in AQP3 extracellular domain. Interestingly, no effect was
observed on AQP1, a strict water channel with no external Cys-residues available for
Auphen binding [2].
The effect of Auphen on AQP3 expressing cells was shown to induce blockage of cell
proliferation [4], thus evidencing a targeted therapeutic effect on cancer types with large
AQP3 expression. The specificity of Auphen towards the aquaglyceroporin subgroup
family was confirmed by detecting its inhibitory effect on AQP7, an aquaglyceroporin
largely expressed in adipocytes [5] and important in adipose tissue homeostasis and
obesity. Our data clearly suggest AQP3 as drug target in cancer cells and point to the
development of aquaporin modulators as therapeutic and diagnostic agents.
References:
[1] Verkman A.S., Anderson M.O., Papadopoulos M.C., Aquaporins: important but elusive drug targets,
Nat Rev Drug Discov, 13, 259-277, 2014.
[2] Martins A.P., Marrone A., Ciancetta A., Galan Cobo A., Echevarria M., Moura T.F., Re N., Casini A.,
Soveral G., Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based
compound, PLoS One, 7, e37435, 2012.
[3] Martins A.P., Ciancetta A., de Almeida A., Marrone A., Re N., Soveral G., Casini A., Aquaporin
inhibition by gold(III) compounds: new insights, ChemMedChem, 8, 1086-1092, 2013.
[4] Serna A., Galan-Cobo A., Rodrigues C., Sanchez-Gomar I., Toledo-Aral J.J., Moura T.F., Casini A.,
Soveral G., Echevarria M., Functional Inhibition of Aquaporin-3 With a Gold-Based Compound
Induces Blockage of Cell Proliferation, J Cell Physiol, DOI 10.1002/jcp.24632, 2014.
[5] Madeira A., de Almeida A., de Graaf C., Camps M., Zorzano A., Moura T.F., Casini A., Soveral G., A
gold coordination compound as a chemical probe to unravel aquaporin-7 function, ChemBioChem,
DOI 10.1002/cbic.201402103, 2014.
INVITED LECTURES
S3 (Elsevier Symposium): Perspectives in lipid and membrane research.
Chairperson: Richard Epand (McMaster University, Ontario, Canada)
IL3.1. The biophysics of lipid signaling during the stress response in plants.
Edgard Kooijmann (Kent University, Ohio, USA)
IL3.2. From the biophysical bench to bedside: understanding phospholipid/protein/cholesterol
cross-talk to produce better therapeutic pulmonary surfactants.
Jesús Pérez-Gil (UCM, Madrid)
IL3.3. Molecular and membrane asymmetry of cardiolipins in mitochondria: signaling
elimination.
Valerian Kagan (University of Pittsburgh, Pennsylvania, USA)
IL3.4. A selection of chemical approaches to lipid research.
Gemma Fabriàs (IQAC, Barcelona)
IL3.5. Relevance of lipid signaling to disease processes.
Richard Epand (McMaster University, Ontario, Canada)
IL3.6. Modulation of membrane curvature by antimicrobial peptides.
Karl Lohner (University of Graz, Austria)
IL3.7. Methods used to determine "oxidative stress" and to rank antioxidants.
Dov Lichtenberg (Tel Aviv University, Israel)
THE BIOPHYSICS OF LIPID SIGNALING DURING
THE STRESS RESPONSE OF PLANTS
Edgard Kooijmann
Kent University, Ohio
In this talk I will detail some of our recent work on the signaling lipid phosphatidic acid,
and its metabolite diacylglycerol-pyrophosphate (DGPP). Diacyglycerol pyrophosphate
an uncommon membrane lipid is found in plants, yeast, and other microorganisms but
never in mammals. DGPP is formed from the membrane lipid phosphatidic acid (PA),
which, in plants, is formed as a response to stressful environmental conditions such as
extreme temperature, salinity, and pathogen attack. The function of the formation of
DGPP during stress is unclear; however, DGPP is unique as it is one of few membrane
lipids with two phosphate groups stacked on top of each other—i.e. a pyrophosphate
group. This pyrophosphate head group is likely crucial to the function of DGPP.
Furthermore, genetic approaches have thus far failed to elucidate the function of DGPP,
other than verify its involvement in stress signaling. Therefore, the physiochemical
properties, specifically the ionization of the head group in the membrane, and the effective
lipid shape were analyzed to elucidate the function of DGPP. These properties can be
compared with those of PA to shed light on the function of this enigmatic lipid.
FROM THE BIOPHYSICS BENCH TO BEDSIDE: UNDERSTANDING
PHOSPHOLIPID/PROTEIN/CHOLESTEROL CROSS-TALK TO PRODUCE
BETTER THERAPEUTIC PULMONARY SURFACTANTS
Jesús Pérez-Gil
Dept. Biochemistry and Molecular Biology, Faculty of Biology,
Complutense University, 28040 Madrid, Spain
Understanding how the lipid/protein complexes of pulmonary surfactant may facilitate
respiratory mechanics in mammalian lungs has taken decades of extensive biophysical
research. Current models propose that surfactant forms highly surface active
phospholipid-rich multilayered films at the respiratory air-liquid interface, to reduce
dramatically surface tension along the successive, very dynamic, compression-expansion
breathing cycles. Phospholipid composition in pulmonary surfactant would have evolved
to sustain coexistence of ordered/disordered lipid phases, and some unusual extremely
hydrophobic proteins, SP-B and SP-C, would be strictly required to ensure proper
membrane-interface traffic of surface-active species. Exogenous clinical surfactants are
designed to mimic these essential structure-function features, and so being useful to
restore surfactant function in respiratory pathologies associated with a lack of operational
surfactant material in the lungs.
However, an important unresolved question is the possible role of cholesterol in
surfactant structure and function. It is clear that the physiological presence of 5-10%
cholesterol with respect to phospholipid mass modulates the lateral structure and the
dynamic behavior of surfactant membranes. On the other hand, comparative biophysical
studies of surfactant from animals exhibiting variable body temperature revealed that
cholesterol may take part of compensatory mechanisms to fit pulmonary surfactant
performance to defined environmental constraints. In contrast, severe respiratory
dysfunctions such as Acute Respiratory Distress Syndrome (ARDS) have been associated
with a defective surfactant and the presence of exacerbated proportions of cholesterol in
the airspaces. As a consequence, cholesterol is usually removed from most clinical
surfactants in use.
Recent evidences that will be summarized suggest that phospholipid and protein
composition in surfactant may have co-evolved with physiological mechanisms that
might use cholesterol to modulate surfactant performance. A proper understanding of
phospholipid/cholesterol/protein interactions is therefore required to design and produce
better clinical surfactants, of utility to treat some of the respiratory pathologies with the
highest mortality and morbidity rates in the critical care units of our hospitals.
MOLECULAR AND MEMBRANE ASYMMETRY OF CARDIOLIPINS
IN MITOCHONDRIA: SIGNALING ELIMINATION
Valerian E. Kagan
Center for Free Radical and Antioxidant Health, Dept. of Environ. Health, Univ. of Pittsburgh,
Pittsburgh, Pennsylvania, USA
kagan@pitt.edu
In contrast to random non-enzymatic chemical reactions of lipid peroxidation, enzyme
catalyzed oxidative modifications of polyunsaturated fatty acyls in phospholipids is
mostly associated with signaling processes. Among the latter, oxidative modification of
cardiolipins in mitochondria are now thoroughly investigated. Cardiolipins [CLs] are
ancient and unusual dimeric phospholipids localized in the plasma membrane of bacteria
and in the inner mitochondrial membrane of eukaryotes.
In this talk, I will document the signaling function of externalized on the surface of
damaged mitochondria CLs as an elimination signal for mitophagy realized through the
recognition by autophagy related microtubule-associated light chain 3 protein. I will
review interactions of CLs with an intermembrane space hemoprotein, cytochrome c [cyt
c], yielding a complex with the peroxidase competence, and present redox reaction
mechanisms of cyt c-catalyzed CLs peroxidation as a required stage in the execution of
apoptosis. Finally, I will illustrate the role of CLs oxidation by cyt c as a new source of
oxygenated lipid mediators.
A SELECTION OF CHEMICAL APPROACHES
TO LIPID RESEARCH
Gemma Fabrias1, José Luis Abad1, Josefina Casas1 and Antonio Delgado1,2
1
Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya
(IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM).
Jordi Girona 18-26, 08034 Barcelona, Spain.
2
Universidad de Barcelona (UB), Facultad de Farmacia, Unidad de Química Farmacéutica (Unidad
Asociada al CSIC), Avda. Joan XXIII s/n, 08028 Barcelona, Spain.
Progress in the understanding of lipid metabolism and functions has benefited from the
contribution of all areas of chemistry, from biophysical to analytical and synthetic organic
chemistry. A selection of examples of different chemical approaches applied to lipid
research is given in this talk, with a special focus of lipidomics and the extension of click
chemistry to lipid biology studies.
Lipidomics aims to analyze cellular lipid pathways and networks by quantitatively and
comprehensively defining the lipidomes of biological sources. Given the depth and
wealth of the structural information desired, mass spectrometry (MS) is the most
preferred method of analysis. The introduction of MS-based lipidomics enabled
biological studies at a previously unattainable level of complexity and allowed
researchers to quantitatively define lipid compositions from many different sources,
ranging from membrane subdomains, vesicles, and viral particles to cells, tissues, and
whole organisms. A future challenge is the development of integrative work flows that
combine different extraction procedures and mass spectrometric approaches to
comprehensively assess all the lipid species in a given sample. Moreover, the
development of software dedicated to lipidomics will enable comprehensive studies of
individual samples and the management of enormous data sets generated by highresolution mass spectrometers.
Click reactions are defined broadly as those that meet the necessary criteria of being
selective, high yielding, and having good reaction kinetics. A subclass of click reactions
whose components are inert to the surrounding biological milieu is termed bioorthogonal.
Bioorthogonal chemical reactions are paving the way for new innovations in biology.
These reactions possess extreme selectivity and biocompatibility, such that their
participating reagents can form covalent bonds within richly functionalized biological
systems. Despite the extensive use of click chemistry in chemical biology, its application
to lipid biology studies is limited. Interesting applications include lipid trafficking studies
and the differential labeling of lipid populations for the mass spectrometry analysis of
pooled samples.
MEMBRANE BIOPHYSICS INTERSECTING BIOLOGICAL FUNCTION
Richard M. Epand
McMaster University, Hamilton, Ontario, Canada
epand@mcmaster.ca
Membrane biophysics encompasses studies both of the bulk material properties of the
membrane as well as studies of the properties and functions of individual molecular
components of membranes. The relationship of membrane proteins that include receptors,
membrane-bound enzymes and transporters to biological function is generally obvious.
The activity of these proteins is often modulated by the bulk properties of the membrane,
so the two aspects of bulk properties and molecular properties, are not completely
independent of each other but are inter-related. Among the bulk properties of the
membrane that have been shown to be important for biological function include
membrane curvature and membrane domain formation. Membrane curvature properties
include both the physical curvature of the membrane as well as the intrinsic curvature.
The differences between the two will be discussed, as well as how they can modulate
protein function. In addition to the mean curvature, there is also Gaussian curvature that
is thought to have an important role in membrane fusion. The other physical property we
will discuss is membrane domain formation. It is generally agreed that biological
membranes are not homogeneous, but there are regions in which the concentration of
certain components is increased. These are referred to as domains, but the nature and
composition of these domains is currently being refined. The particular type of domain
termed a “raft” has attracted particular attention as a possible platform in which signal
transduction proteins can effectively interact with one another. Bilayer lipid asymmetry
is another aspect of the inhomogeneity of a membrane that is associated with membrane
function. Finally, there is the rapidly evolving field of membrane protein structure that
has given many experimental challenges. A wide variety of methods have been applied
to this problem including NMR, X-ray diffraction and electron microscopy imaging. With
the development of new detector methods, there is now a promise of rapid advances in
generating structures of membrane proteins without the need for use of detergents or
crystallization.
Acknowledgements: This work has been sponsored by the Canadian Natural Sciences and
Engineering Research Council, grant 9848.
MODULATION OF MEMBRANE CURVATURE
BY ANTIMICROBIAL PEPTIDES
Karl Lohner
Institute of Molecular Biosciences, Biophysics Division, University of Graz, Austria
Antimicrobial peptides, short amphipathic molecules, represent promising candidates for
novel antibiotics, which are urgently needed owing to the steady increase of pathogenic
bacteria that are multi‐resistant to commercially available antibiotics. Several models
have been suggested to explain their killing activity, which involve interaction with lipid
membranes. Thereby, the property of the polar/apolar membrane interface strongly
depends on the lipid composition, which differs between bacterial and mammalian
cytoplasmic membranes. In general, lipids with molecular shapes different from cylinders
will form monolayers that either curve away or towards the aqueous phase. However in
a planar membrane they are forced into a flat topology leading to significant curvature
elastic stress that is stored within the membrane, which can have several functional
consequences. Hence, the biological activity of antimicrobial peptides may in part be due
to modulation of membrane stored elastic stress, i.e. curvature strain. Latter can be related
to the ratio of lamellar to non-lamellar phase forming lipids present in the membrane
governing the response towards interaction with amphipathic peptides. For example, the
cytoplasmic membrane of Gram-negative bacteria is rich in lipids like PE, exhibiting a
negative spontaneous curvature, and therefore may be more prone to membrane
disruption by such a mechanism than mammalian plasma membranes, which contain a
high amount of bilayer forming lipids. In fact, a number of antimicrobial peptides promote
generation of saddle-splay membrane curvature, which releases the high stored elastic
energy in cytoplasmic membranes. Moreover, insertion of peptides in the membrane
interface will change the lateral pressure profile, which can lead to changes of the
conformational equilibrium of integral membrane proteins and hence to impairment of
membrane function. This may be an additional mechanism to - or a consequence of - the
interfacial activity by which antimicrobial peptides kill bacteria.
METHODS USED TO DETERMINE "OXIDATIVE STRESS"
AND TO RANK ANTIOXIDANTS
Dov Lichtenberg and Ilya Pinchuk
School of Medicine, Tel Aviv University, Israel
physidov@post.tau.ac.il
The term ‘oxidative stress’ (OS) is an ill-defined intuitive term that cannot be described
by a universal criterion. Different criteria of ‘oxidative stress' correlate with each other
only when the results reflect OS determined on the basis of the levels of similar biomarkers (e.g. different DNA fragmentation products or different biomarkers of lipid
peroxidation). Since bio-markers that reflect products of different types do not correlate
with each other, we think that there are different "types" (or Classes) of oxidative stress,
each of which can be estimated on the basis of different methods [Dotan et al, 2004]. The
typical time dependence of peroxidation is sigmoidal, namely rapid peroxidation is
preceded by a “lag phase” that can be used to evaluate the resistance of the lipids to
oxidation. The possibility of evaluating the OS because of the observed sigmoidal shape
of LDL peroxidation is problematic because it requires fractionation of lipoproteins
(particularly due to peroxidation during fractionation).
We have developed a method that can be used to evaluate the OS on the basis of ex-vivo
measurements of peroxidation of lipids in unfractionated serum [Schnitzer et al, 1995]
and we recently analyzed the whole kinetic profile in terms of rate constants and
concentrations based on experimentally attainable factors, particularly well-defined time
points [Pinchuk and Lichtenberg, 2014]. We also looked for available methods to rank
antioxidants and observed that several popular methods are conducted in solution and can
therefore not be regarded relevant to peroxidation in biological systems, which occurs at
lipid-water interphases [Pinchuk and Lichtenberg, 2012& 2014].
We used our optimized assay to (i) assess the susceptibility of fractionated LDL and
HDL as well as of mixtures of these lipoproteins [Raveh et al, 2000], (ii) The OS in
individuals under different pathophysiological conditions, which revealed that different
diseases are associated with different "types" of OS [Dotan et al, 2012]. (iii) We studied
the effects of membrane composition on the susceptibility of oxidizable lipids and on the
effect of antioxidants on lipid peroxidation in relatively simple model membranes
(liposomes). These results will be briefly described and discussed.
INVITED LECTURES
S4: Protein folding, stability, function and dynamics.
Chairperson: Miquel Pons (UB, Barcelona)
IL4.1. Protein folding, ions and regulation.
Javier Sancho (Unizar, Zaragoza)
IL4.2. Looking at inborn errors from a structural viewpoint: some examples around
urea cycle disorders.
Vicente Rubio (IBV, CSIC, Valencia)
IL4.3. Prediction of hydrodynamic and other solution properties of partially
disordered proteins.
José García de la Torre (UM, Murcia)
IL4.4. Modelling and computer simulation of ion channels.
Carmen Domene (King's College London, UK)
PROTEIN FOLDING, IONS AND REGULATION
Javier Sancho
Universidad de Zaragoza, Zaragoza, Spain
jsancho@unizar.es
Protein folding is an interesting and partly understood phenomenon. Fortunately, after
decades of biophysical investigation, it has gathered renewed interest with the realization
that most non-infectious diseases are probable protein folding diseases. On the other hand,
the reversibility of the folding reaction makes it suitable for cellular regulation, which
might prove an active field of research in the future. One easy way for proteins to undergo
reversible folding/unfolding is through their interaction with ions. Eukaryotic cells take
indeed great effort to maintain different ion concentrations in their different
compartments. Acidification of organelles, such as endosomes or lysosomes has been
sometimes associated to degradative functions or to ways of modifying protein/protein
interactions. I propose that organelles such as the endosome might use changes is the
concentrations of several ions, e.g. Ca++ or H+, to induce the unfolding of receptors as a
drastic way to reduce their affinity for their cargos and allow an easy delivery. Receptors
that are recycled to the cell surface where the key ionic concentrations are restored to
initial values can then refold and recover their capability of binding more cargo. This
hypothesis will be illustrated with studies of the LDL receptor, a modular calcium binding
protein that binds circulating LDL and VLDL at the cell surface and transport them to the
endosome where they are degraded, while the receptor is recycled back to the cell surface.
Mutations in the LDLR, many of them point mutations, are involved in a disease called
Familiar Hypercholesterolemia. I will also show that these mutations seem to interfere
with either cargo binding or with the ability to experience ion-induced reversible folding.
Acknowledgements: We acknowledge financial support, among others, from grants BFU2010-16297,
Spain and Grupo Protein Targets B89 from the Diputación General de Aragón (Spain).
References
[1] X. Arias-Moreno, A. Velazquez-Campoy, JC Rodríguez, M. Pocoví, J. Sancho, The mechanism of
LDL release in the endosome: Implications of the stability and Ca++ affinity of the fifth binding
module of the LDL receptor, J. Biol. Chem., 283, 22670-22679, 2008.
[2] X. Arias-Moreno, S. Cuesta-Lopez, O. Millet , J. Sancho, A. Velazquez-Campoy,Thermodynamics of
Protein-Cation Interaction: Ca+2 and Mg+2 Binding to the Fifth Binding Module of the LDL Receptor,
Proteins, 78, 950–961, 2010.
[3] J. Martínez-Oliván, X. Arias-Moreno, A. Velazquez-Campoy, O. Millet, J. Sancho, LDL
receptor/lipoprotein recognition: endosomal weakening of apo B and apo E binding to the convex
face of the LR5 repeat. FEBS J., 281, 1534-1546, 2014.
[4] J. Martínez-Oliván, Z. Rozado-Aguirre, X. Arias-Moreno, V. E. Angarica A. Velazquez-Campoy, J.
Sancho, LDL receptor is a calcium/magnesium sensor: Role of LR4 and LR5 ion interaction kinetics
in LDL release in the endosome. FEBS J., 2014, In Press DOI:10.1111/febs.12811.
LOOKING AT INBORN ERRORS FROM A STRUCTURAL VIEWPOINT:
AN EXAMPLE AMONG UREA CYCLE DISORDERS
Sergio de Cima1, , Luis Mariano Polo1, Carmen Díez-Fernández1,
Javier Cervera1, Ignacio Fita2, Vicente Rubio1
1
Instituto de Biomedicina de Valencia of the CSIC, and Group 739 of the Centro de Investigación
Biomédica en Red sobre Enfermedades Raras (CIBERER)
del Instituto de Salud Carlos III, Valencia, Spain
rubio@ibv.csic.es
2
Institut de Biologia Molecular de Barcelona (CSIC), Barcelona, Spain
The urea cycle is used by mammals to get rid of the ammonia derived from protein catabolism,
which is highly neurotoxic. Carbamoyl phosphate synthetase 1 (CPS1), a large (1462 residues)
six-domain protein catalyzing a complex three-step reaction involving two homologous but
separate phosphorylation centers, is a key catalyst and controller of the urea cycle and is a very
abundant (20%) protein in the matrix of liver mitochondria. A paramount trait of this enzyme is
its absolute requirement for N-acetyl-L-glutamate (NAG), an allosteric activator without which it
is inactive, which is used an on/off switch to prevent ammonia depletion. The report of CPS1
regulation by multiple lysine acylation and by deacylation by sirtuin 5 connected the urea cycle
with the age-control machinery (Nakagawa et al. Cell 2009; 137:560). CPS1 deficiency (CPS1D)
is a urea cycle disorder causing severe neonatal hyperammonemia leading to mental retardation
or even to death. >300 mutations have been reported in CPS1D patients, of which the majority
are missense mutations showing little recurrence and having unproven disease-causing potential.
It was impossible either to utilize expression studies or to make detailed structural inferences to
clarify the mutation's effects. Only the structure of Escherichia coli CPS was known, but this
enzyme has limited sequence similarity with CPS1, it is active in the absence of effectors, not
being activated by NAG, and it uses glutamine as the internal ammonia source, whereas CPS1
cannot use glutamine and utilizes as substrate ammonia with high affinity. Using a
baculovirus/insect cell system we have finally succeeded in producing recombinant human CPS1
in large amount and pure form. This has opened the way to experimental examination of the
mutations' effects, as exemplified in our recent study (Díez-Fernández et al. Mol Genet Metab
2014; in press) with the reported mutations affecting a CPS1 domain for which we have identified
the functions, ascertained the mutations' disease-causing potential and clarified the reasons for
the high mutational eloquence of the domain. Determination of the CPS1 structure also appeared
essential for understanding CPS1 function and its control by the NAG switch and by acylation,
and to rationalize and even to predict the pathogenicity of the CPS1 clinical mutations without
having to express them. Thanks to the recombinant production of human CPS1, we have now
crystallized this enzyme and determined its structure at 2.4 Å-resolution, in apo and ligand-bound
(NAG and ADP/Pi) forms. The liganded structure revealed how does NAG bind in a pocket of
the C-terminal domain and has identified elements that are stabilized by ADP binding, as well as
conformational changes induced by NAG and ADP binding that lead to define the carbamate
tunnel, which in the apo form is heavily branched and open to the environment.
Our structures decipher the CPS1 inability to use glutamine and reveal a potential channel for
ammonia intake. Furthermore, they help rationalize the disease-causing role of most clinical CPS1
mutations.
Supported by Fundación Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish
(BFU2011-30407; FPU to CD-F) governments.
PREDICTION OF HYDRODYNAMIC AND OTHER SOLUTION PROPERTIES
OF PARTIALLY DISORDERED PROTEINS
Diego Amorós, Álvaro Ortega, José García de la Torre
Departamento de Química Física, Facultad de Química, Campus Regional de Excelencia International
“Campus Mare Nostrum”, University of Murcia, E-30071 Murcia, Spain
jgt@um.es
Intrinsically disordered (ID) proteins are of great present importance for their recently
discovered role in a number of physiological processes. The availability of tools for
predicting their solution properties, which could be used in the analysis of experimental
data, in order to ascertain their structural and dynamics properties, is an evident need.
Such tools are now well developed for ordered, and particularly quasirigid proteins. We
have intended to develop a systematic computational scheme for partially disordered
proteins [1], which is a subgroup of ID proteins characterized for having well defined and
ordered nearly rigid domains, and highly disordered, flexible linkers or tails. The
consideration of flexibility in hydrodynamic calculations [2] is now feasible with the help
of the SIMUFLEX software [3], which predicts the solution conformation and dynamics
of models with various kinds and degrees of flexibility from Brownian dynamics or
Monte Carlo simulations.
We have applied this methodology to various partially disordered proteins, predicting
hydrodynamic properties (sedimentation and diffusion coefficients, NMR relaxation
times) and scattering-related ones (radius of gyration, SAXS/SANS intensities and
distribution of distances), with remarkable success. Several biomolecular atomic-level
modelling tools are used to construct coarse-grained models that can be treated with our
simulation programs. Calculated values of single-valued properties, like the
sedimentation coefficient and the radius of gyration agree very well (differences of a few
percent, in most cases), with the experimental values, within their range of experimental
error. Even the whole scattering and distance distribution diagrams show a satisfactory
agreement with the measured ones. Thus, we have developed tools, established protocols
and tested a complete methodology that would permit the use of the classical techniques
of characterization for the elucidation or validation of structures of intrinsically
disordered proteins.
Acknowledgment. Supported by grants CTQ2012-33717 (Ministry of Economy and Competitiveness),
including FEDER funds, and 04486/GERM/06 (Fundacion Seneca).
[1] D. Amorós, A.Ortega, J. Garcia de la Torre “Prediction of hydrodynamic and other solution properties
of partially disordered proteins with a simple, coarse-grained model”. J. Chem. Theor. Comput., 9,
1678-1685 (2013).
[2] J. García de la Torre, et al. “Methods and tools for the prediction of hydrodynamic coefficients and
other solution properties of flexible macromolecules in solution. A tutorial minireview”, Macromol.
Biosci., 10, 721-730 (2010).
[3] J. García de la Torre, et al “SIMUFLEX: Algorithms and tools for simulation of the conformation and
dynamics of flexible molecules and nanoparticles in dilute solution”, J. Chem. Theory Comput. 5,
2606-2618 (2009).
MODELLING AND COMPUTER SIMULATION OF ION CHANNELS
Carmen Domene
Department of Chemistry, King’s College London, UK
carmen.domene@kcl.ac.uk
Recently, there has been an enormous progress in the broad application of computation
to topical problems in areas ranging from chemistry and soft matter, to the interface
between physical science and biomedical research. By means of selected examples from
the work I have carried out, I will illustrate the current status of the field that employs
computer simulation methodologies, and provide an overview of the current knowledge
we have about permeation, selectivity and gating in ion channels, an important family of
membrane proteins.
INVITED LECTURES
S5: Protein Structure.
Chairperson: Juan A. Hermoso (IQFR, CSIC, Madrid)
IL5.1. Crystal structure of RNA polymerase I.
Carlos Fernández Tornero (CIB, CSIC, Madrid)
IL5.2. Biophysics of the assembly of RNA stress granules: The Pub1-eIF4G1 case.
José M. Pérez-Cañadillas (IQFR, CSIC, Madrid)
IL5.3. Visual protein photoreceptors: misfolding, stability and retinal disease.
Pere Garriga (EET-UPC, Terrassa, Barcelona)
IL5.4. Structure and regulation of a non-canonical RNA-dependent RNA polymerase.
Núria Verdaguer (IBMB, Barcelona)
CRYSTAL STRUCTURE OF RNA POLYMERASE I
Carlos Fernández Tornero
Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
In eukaryotes ribosome biosynthesis starts with ribosomal RNA production by RNA
polymerase I (Pol I), a process that is critical to regulate cell growth and proliferation.
We were able to obtain the crystal structure of yeast Pol I, a 14-subunit complex with a
total mass of 590 kDa, at 3.0 Å resolution [1]. The current communication will describe
the functional implications unveiled by this wonderful structure.
The structure represents the latent state of the enzyme, characterized by three major
features. First, it forms dimers that involve the C-terminal tail of the stalk subunit A43.
Second, the two enzyme halves pivot along the DNA-binding cleft to produce an open
cleft and an unfolded bridge helix. Third, an extended loop in subunit A190 mimics the
DNA backbone along the cleft, hampering nucleic acid binding. All three features must
be resolved during enzyme activation.
The Pol I crystal structure also reveals intrinsic modules that only bind transiently in other
RNA polymerases. Subunit A12.2 inserts a TFIIS-like zinc ribbon into the active site,
providing insight into its role in RNA cleavage and Pol I insensitivity to α-amanitin. The
A49-A34.5 heterodimer binds the outer side of subunit A135 through a TFIIF-like
dimerization module, suggesting how this heterodimer may function at different stages
of rRNA synthesis.
Crystal structure of RNA polymerase I
Reference:
[1] C. Fernández-Tornero, M. Moreno-Morcillo, U.J. Rashid, N.M.I. Taylor, F.M. Ruiz, T. Gruene, P.
Legrand, U. Steuerwald, C.W. Müller, “Crystal structure of the 14-subunit RNA polymerase I”, Nature,
502, 7473, 644–649, 2013.
BIOPHYSICS OF THE ASSEMBLY OF RNA STRESS GRANULES:
THE Pub1-eIF4G1 CASE
Santiago Martínez-Lumbreras1,2 and José Manuel Pérez-Cañadillas1
Departamento de Química-Física Biológica, Instituto de Química-Física “Rocasolano”, CSIC.
c/ Serrano 119, 28006 Madrid, Spain.
jmperez@iqfr.csic.es
2
Present address: Department of Chemistry, School of Biomedical Science, King’s College London.
7 Trinity Street, London SE1 1DB, United Kingdom
1
Living cells have developed post-transcriptional mechanisms of rapid response against
different types of environmental stress. These involve translational arrest through mRNA
localization in cytoplasmic granular-like structures such as stress granules and P-bodies.
The nucleation of these sub-cellular assemblies is mediated by amyloid-like sequences
that are present in some RNA binding proteins and translation initiation factors. Pub1 and
Tif4631 are essential proteins of many types of stress granules in yeast. Here we describe
the structures and interaction modes of Pub1 and Tif4631 as determined by NMR. The
C-terminal RRM domain of Pub1 contains a novel N-terminal helix, exclusive of Pub1
and vertebrate homologs, that we named “TIA-1 C-terminal domain-like RRM” (TRRM).
Pub1 TRRM recognizes U-rich RNA but more importantly interacts with the N-terminal
region of Tif4631 using a surface compatible to the RNA binding one. Tif4631 Nterminal domain (about 400 residues) is largely unstructured but contains several MoRFs
(Molecular Recognition Fragments) involved in intramolecular interactions, RNA
binding and protein-protein recognition (Pub1 and Pab1). Pub1 TRRM targets at least
two these MoRFs and under certain circumstances aggregates upon binding. The structure
of a chimeric construct between the two proteins reveals some of the molecular details of
this weak interaction. Tif4631 N-ter domain is chemically unstable (autoproteolysis and
deamidation) in a pH dependent manner and evolves to the formation of hydrogels;
probably amyloid-like. These results are integrated in a model that pretends to explain the
biophysical bases of the stress granule formation in yeast.
VISUAL PROTEIN PHOTORECEPTORS: MISFOLDING,
STABILITY AND RETINAL DISEASE
Pere Garriga, Sundaramoorthy Srinivasan, Xiaoyun Dong,
Miguel Fernández, Margarita Morillo, Eva Ramon
Secció de Terrassa, Departament Enginyeria Química, Universitat Politècnica de Catalunya,
Edifici Gaia, Rambla San Nebridi 22, 08222 Terrassa, Spain
pere.garriga@upc.edu
Rhodopsin and cone opsins, the visual pigments of the vertebrate retina, are a distinct
group of G-protein-coupled receptors (GPCRs) which are responsible for light capture in
the first molecular event of the complex process of vision. These receptors are covalently
bound to their natural ligand, the chromophoric 11-cis-retinal, in a structurally versatile
binding pocket that allows their conformational stabilization and functional activation.
Various retinal analogs were reported to behave as chromophores for these retinal
receptors but with different specific physical properties and activation abilities.
Rhodopsin -the first GPCR to be crystallized and whose three dimensional structure was
elucidated- has served as the prototypical model for class A GPCRs for more than a
decade. Upon photoactivation, rhodopsin binds and activates the specific heterotrimeric
G-protein transducin. The details of this light-dependent activation, including the
molecular interaction with transducin, have not been fully elucidated. Mutations in
rhodopsin have been associated with the retinal degenerative disease retinitis pigmentosa
(RP). The folding, degradation and aggregation of some of these RP mutant rhodopsins can
be manipulated by drugs or molecular chaperones [1]. Our goal is to deepen our knowledge
of the molecular consequences of such mutations (naturally-occurring and newly
designed) while gaining, at the same time, new insights into the structural requirements
of the receptor activation process. We are also interested in the effect of lipids and metal
ions on the structure, stability and function of these receptors. The ligand-protein
interactions comparison between rhodopsin and cone opsin pigments, as a model for
GPCRs, is also explored. We will describe our recent results on rhodopsin mutations
associated with RP or potentially relevant for visual pigment evolution, the effect of
docosohexaenoic acid lipid on the stability of rhodopsin [2], and the structural differences
between rhodopsin and red cone opsin with regard to ligand binding after photoactivation
[3]. Overall, our studies enhance our understanding of the molecular mechanisms of
visual phototransduction, the biology of GPCRs in general, and importantly, they may
help develop potential approaches to treat RP caused by rhodopsin mutations.
Acknowledgements: Grants SAF2011-30216-C02-01 from MICINN, CIVP16A1861 from Fundación
Ramón Areces, and Grups de Recerca Consolidats de la Generalitat de Catalunya (2009 SGR 1402).
References
[1] Aguilà M, Bevilacqua D, McCulley C, Schwarz N, Athanasiou D, Kanuga N, Novoselov S, Lange
CAK, Ali R, Bainbridge JW, Gias C, Coffey PJ, Garriga P, and Cheetham ME. Hsp90 inhibition
protects against inherited retinal degeneration. Human Molecular Genetics 23, 2164-2175 (2014)
[2] Sánchez-Martín MJ, Ramon E, Torrent-Burgues J and Garriga P. Improved conformational stability
of the visual G-protein coupled receptor rhodopsin by specific interaction with docosahexaenoic acid
phospholipid. ChemBioChem 14, 639-644 (2013)
[3] Srinivasan S, Ramon E, Cordomí A, and Garriga P. Binding specificity of retinal analogs to
photoactivated visual pigments suggest mechanism for fine-tuning GPCR-ligand interactions.
Chemistry and Biology 21, 369-378 (2014)
STRUCTURE AND REGULATION OF A NON-CANONICAL RNADEPENDENT RNA POLYMERASE
Diego Ferrero1,2, José F. Rodríguez2, Núria Verdaguer1
1
Institut de Biología Molecular de Barcelona, CSIC, Parc Científic de Barcelona,
Baldiri i Reixac 15, 08028-Barcelona, Spain
nvmcri@ibmb.csic.es
2
Centro Nacional de Biotecnología, CSIC, Darwin nº 3, 28049-Madrid. Spain
RNA viruses include most of the etiological agents of emerging or re-emerging diseases
affecting humans and animals and, in consequence, a constant burden on our healthcare
systems and economy. One strategy to limit the impact of RNA viruses is to prevent their
replication, therefore, a deep knowledge of the mechanisms by which these pathogens
replicate and transcribe their genetic material is essential. All RNA viruses encode at least
one protein involved in nucleic acid synthesis, the RNA-dependent RNA polymerase
(RdRP). RdRPs belong to a superfamily of template-directed nucleic acid polymerases,
including DNA-dependent DNA polymerases (DdDP), DNA-dependent RNA
polymerases and reverse transcriptases (RT). All theses enzymes share a cupped right
hand structure -including fingers, palm and thumb domains-, catalyze phosphodiester
bond formation through a conserved two-metal ion mechanism and display similar kinetic
mechanism. Unique to RdRPs is the “closed-hand” conformation, in opposition to the
“open-hand” found in other polynucleotide polymerases [1]. RdRP amino acid sequences
are highly variable, however, strong amino acid conservation can be observed in regions
that are directly involved in RNA and nucleotide selection, binding and catalysis. The
prototypic viral RdRPs possess seven of such regions or motifs arranged in the order
G,F,A,B,C,D and E from amino- to carboxy- terminus. The only exceptions to this
scheme were found in the double-stranded RNA (dsRNA) Birnaviruses and in the
positive-sense, single-stranded RNA (ssRNA) Permutotetraviruses, where the palm motif
C is encoded upstream of motif A [2, 3]. In this talk we will discuss the recent results
obtained in the structural characterization of the RdRP domain of the Permutotetravirus
Thosea asigna virus (TaV). The structure shows that despite the permuted connectivity,
the TaV RdRP palm maintains the geometry of the catalytic residues found in canonical
polymerases. In addition, biochemical and structural data allowed the identification of
two essential elements, regulating the polymerization activity.
Acknowledgements: This work has been sponsored by Spanish MINECO, project BIO2011-24333,
and by the SILVER Cooperation project GA No. 260644 of the EU.
References
[1] Cristina Ferrer-Orta, Armando Arias, Cristina Escarmis, NuriaVerdaguer, N. A comparison of viral
RNA-dependent RNA polymerases. Curr Opin Struct Biol, 16, 27-34, 2006.
[2] Alexander E Gorbalenya, et al. The palm subdomain-based active site is internally permuted in viral
RNA-dependent RNA polymerases of an ancient lineage. J Mol Biol, 324, 47-62, 2002.
[3] Damià Garriga, Aitor Navarro, Jordi Querol-Audi, Fernando Abaitua, José F Rodriguez, Nuria
Verdaguer. Activation mechanism of a noncanonical RNA-dependent RNA polymerase. Proc Natl
Acad Sci U S A 104, 20540-5. 2007.
INVITED LECTURES
S6: Cell biophysics.
Chairperson: María García Parajo (ICFO, Barcelona)
IL6.1. Optopharmacology to regulate endogenous proteins with light.
Pau Gorostiza (IBEC, Barcelona)
IL6.2. Synaptic dysfunction and nerve terminal degeneration.
Rafael Fernández-Chacón (IBiS/HUVR/CSIC/US/CIBERNED, Sevilla)
IL6.3. The disordered boundary of the cell: Intrinsically disordered proteins on cell
membranes.
Miquel Pons (UB, Barcelona)
IL6.4. Looking at membrane fluctuations in red blood cells: dynamics of the spectrin
cytoskeleton.
Francisco Monroy (UCM, Madrid)
OPTOPHARMACOLOGY TO REGULATE ENDOGENOUS PROTEINS
WITH LIGHT
Pau Gorostiza
Catalan Institution for Research and Advanced Studies (ICREA)
Institute for Bioengineering of Catalonia (IBEC)
Network Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)
pau@icrea.cat
Most biological processes are mediated by protein-protein interactions (PPIs), which have
emerged as important pharmacological targets. Peptide inhibitors with nanomolar
affinities were recently developed and demonstrated anticancer activity in vivo. In order
to dissect the precise orchestration of PPIs in cells, it would be very useful to complement
the pharmacologic selectivity of inhibitors with a means of remotely controlling their
kinetics and site of action. Here, we present a peptide design and screening method to
obtain photoswitchable inhibitors of protein-protein interactions (PIPPIs), and we apply
it to photo-regulate clathrin-mediated endocytosis (CME) in living cells. The selective
manipulation of CME with light using these peptides, named Traffic Lights (TL) because
they act as stop & go signals for membrane traffic, constitutes a novel tool to control cell
signaling in spatiotemporally defined patterns. TL peptides can be applied to dissect the
role of CME in complex cellular functions like receptor internalization, cell growth,
division and differentiation.
PRESYNAPTIC DYSFUNCTION AND NEURODEGENERATION
IN THE ABSENCE OF A SYNAPTIC VESICLE CO-CHAPERONE:
WHAT HAPPENS BEYOND THE NERVE TERMINALS?
Rafael Fernández-Chacón
Instituto de Biomedicina de Sevilla (IBiS, Hosp.Univ. Virgen del Rocío/CSIC/Universidad de Sevilla),
Dpto. de Fisiología Médica y Biofísica and CIBERNED, Sevilla, Spain
rfchacon@us.es
Nerve terminals are able to maintain the continuous release of neurotransmitters during
extended periods of time at locations far away from the cell soma. For example,
presynaptic terminals from tonic motorneurons receive from 300.000 to 500.000 action
potentials per day (Hennig and Lomo, Nature 1985) imposing on SNARE complexes a
heavy-duty cycling of protein folding and unfolding reactions. Cysteine String Proteinalpha (CSP-alpha) is a synaptic vesicle protein that, together with Hsc-70 and SGT (small
glutamine-rich protein), forms a chaperone complex essential to maintain a functional
pool of SNAP25 and to promote SNARE complex assembly (Chandra et al., Cell 2005;
Sharma et al. Nat. Cell Biol. 2011). Interestingly knock-out mice lacking CSP-alpha
suffer from early lethality due to presynaptic degeneration (Fernández-Chacón et.al.,
Neuron 2004). We have recently found that motorneurons require CSP-alpha to maintain
the readily releasable vesicular pool and synaptic vesicle recycling (Rozas., et al., Neuron
2012). Interestingly, in central neurons, we have shown that CSP-alpha prevents activitydependent degeneration of GABAergic synapses in high firing rate parvalbumin-positive
neurons, indicating that high-neural activity increases synapse vulnerability and CSPalpha is essential to maintain presynaptic function under a physiologically high-activity
regime (García-Junco-Clemente et al., JNeurosci. 2010). In my talk I will discuss recent
unexpected findings that uncover a deregulation of adult neurogenesis at the dentate gyrus
in the brain of CSP-alpha KO mice. We think that knock-out mice lacking CSP-alpha
offer a particularly useful model to investigate how neural circuits react to progressive
presynaptic dysfunction and neurodegeneration. In addition, our results might reveal
unanticipated functions of CSP-alpha beyond the maintenance of synaptic vesicle
trafficking at the nerve terminals.
Supported by Ministerio de Economía y Competitividad (BFU2010-15713), Junta de Andalucía (P12CTS-2232), Instituto de Salud Carlos III and FEDER.
THE DISORDERED BOUNDARY OF THE CELL:
INTRINSICALLY DISORDERED PROTEINS ON CELL MEMBRANES
Anabel-Lise Lerroux1,3, Borja Mateos, 1 Miguel Arbesú 1, Alba Pulido,2 Enric Mayans,2
Jordi Ignés-Mullol,2 Josep Claret,2 Francesc Sagués,2 and Miquel Pons1
1
Biomolecular NMR Laboratory, Department of Organic Chemistry,
University of Barcelona, Barcelona, Spain.
mpons@ub.edu
2
Department of Physical Chemistry, University of Barcelona, Barcelona, Spain.
2
Institute for Research in Biomedicine (IRB), Barcelona, Spain.
A number of signaling proteins are attached to the surface of the cell membrane by an
anchoring unit connected to the main body of the protein by an intrinsically disordered
region. The Src family of kinases constitutes a paradigmatic case in which the regulatory
SH3 and SH2 domains and the kinase domain are linked to the membrane anchoring
myristoylated SH4 domain through the intrinsically disordered Unique domain (UD). We
shall present on going work on the characterization of the myristoylated Unique domain
of Src, alone (MyrUsrc), fused to the SH3 domain 8murUSH3) or fused to Green
Fluorescent Protein (MyrUGFP).
Myristoylation was achieved either in vivo, by co-expression with N-myrystoyl
transferase (NMT) or in vitro by reaction of purified protein with MyrCoA in the presence
of NMT. Monolayers formed by pure myristoylated proteins or by protein-lipid mixtures
were characterized by Langmuir-Blodget techniques. The interaction of myristoylated
proteins with immobilized liposomes was characterized by surface plasmon resonance
(SPR). 15N-labelled myristoylated UD bound to sonicated lipids vesicles was studied by
solution NMR.
The NMR experiments clearly show that the myristoylated UD interacts with membranes
through both the myristoylated SH4 domain and an internal Unique Lipid Binding
Region. SPR experiments show that the myristoylated UD constructs bind reversibly to
immobilized liposomes. Lagmuir-Blodget experiments show that the pure myristoylated
constructs can form monolayers at the air-water interface and can also be insert onto lipid
monolayers. The system formed by MyrUGFP inserted into dimyristoyl
phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) shows
hysteresis cycles when subjected to compression and expansion. Our results suggest that
the UD plays an active role modulating the interaction of Src with lipid membranes.
Acknowledgements: This work has been sponsored by the Spanish MINECO (BIO2010-15683), la
Fundació Marató TV3 and a fellowship from La Caixa to A-LL.
Reference
[1] Perez, Y., Maffei, M., Igea, A., Amata, I., Gairí, M., Nebreda, A.R., Bernadó, P. Pons, M. “Lipid
binding by the unique and SH3 domains of c-Src suggests a new regulation mechanism” Scientific
Reports 3, 1295; DOI:10.1038/srep01295 (2013).
LOOKING AT MEMBRANE FLUCTUATIONS IN RED BLOOD CELLS:
DYNAMICS OF THE SPECTRIN CYTOSKELETON.
Michael Mell, Ruddi Rodríguez-García and Iván López-Montero and Francisco Monroy
Departamento de Química Física I, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain
Biofísica Traslacional, Instituto de Investigación Hospital Doce de Octubre (i+12), 28041 Madrid, Spain
The physiological function of erythrocytes is directly connected to their outstanding
ability to deform in passing through narrow capillaries. In the recent years, experimental
evidence of enhanced cell shape fluctuations related with metabolically-driven activity of
the erythroid cytoskeleton has been increasingly accumulated. However, no direct
observation of the active cytoskeleton forces has been reported so far.
In this talk, we show direct evidence of the active force driving enhanced membrane
fluctuations in human erythrocytes. Both, theoretical and simulation, stochastic analyses
provide evidence for a correlated, ATP-dependent, direct force exerted by molecular
complexes at the cytoskeleton nodes. In addition to active stresses, these metabolic
processes produce effective membrane softening, a mechanical attribute possibly related
with the functional erythroid deformability.
Acknowledgements: This work was supported by MINECO under grants FIS2009-14650-CO2-01 and
FIS2012-35723
INVITED LECTURES
S7: Biophysics of nucleic acids.
Chairperson: Ana I. Azuaga (Ugr, Granada)
IL7.1. The DNA repair machinery: connections and targets.
Jean Marc Egly (IGBMC, Illkirch, France)
IL7.2. Structure and energetics of protein-RNA interactions: new insights from
docking predictions.
Juan Fernández-Recio (BSC, Barcelona)
IL7.3. Fluorescence spectroscopy and microspectroscopy analysis of protein-DNA
interactions involved in the regulation of essential bacterial processes.
Silvia Zorrilla (CIB, CSIC, Madrid)
IL7.4. NMR study of the effect of C2́'-substituted furanoses on the structure and
stability of nucleic acids.
Nerea Martín-Pintado (IQFR, CSIC, Madrid)
NER FACTORS AND ANTITUMOR DRUGS
Jean-Marc Egly
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/Inserm/ULP,
67404 Illkirch Cedex, C. U. Strasbourg, France
One major challenge in cancer therapeutics is to characterize the altered biochemical
pathway (here DNA repair) in the tumor cell that can be selectively targeted by drugs. In
collaboration with PharmaMar, we are investigating the power of several aromatic drugs
that target DNA. Trabectedin, a tetrahydroisoquinoline alkaloid that forms a covalent
bond with the amino group of a guanine in selected triplets of DNA duplexes, was shown
to give rise to double-strand breaks. We demonstrated that the breaks were performed by
the XPF/ERCC1 nuclease, one of the nucleotide excision repair (NER) factors, on the
strand opposite to that bonded by the drug. This resulted in inhibition of RNA synthesis
by preventing binding of transcription factors to DNA, and arresting elongating RNA
polymerase II. We also have demonstrated that trabectedin homologs induced RNA pol
II degradation thus preventing further RNA synthesis
STRUCTURE AND ENERGETICS OF PROTEIN-RNA INTERACTIONS:
NEW INSIGHTS FROM DOCKING PREDICTIONS
Laura Pérez-Cano, Miguel Romero-Durana and Juan Fernández-Recio
Barcelona Supercomputing Center, Barcelona, Spain
juanf@bsc.es
In the last decade, genomics and proteomics efforts have contributed to the description of
the inventory of macromolecules involved in cellular processes. The challenge now is to
understand their interactions, among which we can highlight those involving RNA
molecules and RNA-binding proteins (RBPs). Computational modeling can complement
experimental efforts in understanding the mechanisms of protein-RNA recognition and
the determinants of their specificity. We previously developed OPRA, based on statistical
potentials, to identify RNA-binding sites in proteins [1], which was successfully applied
to understand interesting aspects of RNA-binding mechanism in human translin [2,3].
Now, we are extending current computational approaches for structural modeling of
protein interactions to the prediction of protein-RNA complexes. For that, we have
evaluated different knowledge- and energy-based scoring functions for protein-RNA
docking using a recent benchmark [4]. We used both the bound and unbound states in
order to find out the importance of each scoring term and the role of conformational
flexibility. Structural complementarity was found to be much more relevant for proteinRNA than for protein-protein docking, while desolvation showed the contrary effect.
Interestingly, electrostatics played a more important role in difficult protein-RNA cases.
The strategies developed by proteins to bind RNA show an interesting dependence on the
flexibility of the interacting molecules, which give insights on the protein-RNA binding
mechanism and provide hints for the development of efficient protein-RNA prediction
methods.
Acknowledgements: This work has been sponsored by grant number BIO2010-22324 from Plan
Nacional I+D+i (Spanish Ministry of Science).
References
[1] Laura Pérez-Cano, Juan Fernández-Recio, “OPRA: a propensity-based method to identify RNAbinding sites on proteins”, Proteins, 78, 25-35, 2010.
[2] Elad Eliahoo, Ron Ben Yosef, Laura Pérez-Cano, Juan Fernández-Recio, Fabian Glaser, Haim Manor,
“Mapping of interaction sites of the Schizosaccharomyces pombe protein translin with nucleic acids
and proteins: a combined molecular genetics and bioinformatics study”, Nucleic Acids Research, 38,
2975-2989, 2010.
[3] Laura Pérez-Cano, Elad Eliahoo, Keren Lasker, Haim J. Wolfson, Fabian Glaser, Haim Manor, Pau
Bernadó, Juan Fernández-Recio, “Conformational transitions in human translin enable nucleic acid
binding”, Nucleic Acids Research, 41, 9956-9966, 2013.
[4] Laura Pérez-Cano, Brian Jiménez-García, Juan Fernández-Recio, “A protein-RNA docking benchmark
(II): extended set from experimental and homology modeling data”, Proteins, 80, 1872-1882, 2012.
FLUORESCENCE SPECTROSCOPY AND MICROSPECTROSCOPY
ANALYSIS OF PROTEIN-DNA INTERACTIONS INVOLVED IN THE
REGULATION OF ESSENTIAL BACTERIAL PROCESSES
Silvia Zorrilla1, Carlos Alfonso1, Nathalie Declerck2,
Begoña Monterroso1, Catherine Royer2 and Germán Rivas1
1
Centro de Investigaciones Biológicas. CSIC. Madrid. Spain.
silvia@cib.csic.es
2
Centre de Biochimie Structurale. CNRS, INSERM, Univ. Montpellier. France
Protein-DNA interactions control and regulate central biological processes in bacteria
including sugar metabolism and cell division. Understanding the molecular mechanisms
underlying these essential processes requires a detailed knowledge of the physical
parameters governing the protein-DNA interactions involved. Fluorescence spectroscopy
methods allow determining the affinity, stoichiometry and cooperativity of nucleoprotein
complexes with high sensitivity. In particular, by using fluorescence fluctuation
spectroscopy methods like FCS and FCCS these complexes, often of high affinity, can be
studied with single molecule detection. We have applied these methodologies in
combination with other biophysical tools such as analytical ultracentrifugation to
characterize the protein-DNA interactions determining the regulation of the transcription
of gluconeogenic and glycolytic genes in Bacillus subtilis by the repressors CcpN and
CggR. The affinity and stoichiometry of the complexes of CggR with its operator DNA
were addressed as well as the modulation of these parameters by specific ligands. The
complexes of CcpN with two of its target DNAs, on the other hand, were also
characterized and, as a result of these studies, a new procedure based on FCCS for the
determination of the stoichiometry of protein-DNA complexes was proposed. A similar
combined biophysical strategy was followed to analyze the nucleoprotein complexes of
SlmA, a sequence specific DNA binding protein that participates in the control of the
positioning of the division ring in E coli by preventing its assembly nearby the
chromosome. The complexes of this protein with different target DNA sequences were
characterized and the effect of such complexes on the polymerization of FtsZ, the main
bacterial division protein, was addressed. Through these examples, we show how the
detailed biophysical analysis of the interactions of DNA binding proteins allows getting
some insight into the functional mechanisms of the key bacterial processes in which they
participate.
Acknowledgements: This work has been sponsored by the Spanish Government through Grant
BIO2011-28941-C03 and by EC through grant MERGT-CT-2006-046474
NMR STUDY OF THE EFFECT OF C2’-SUBSTITUTED FURANOSES
ON THE STRUCTURE AND STABILITY OF NUCLEIC ACIDS
Nerea Martín-Pintado1, Ramón Campos-Olivas2, Guillem Portella3,
Modesto Orozco3, Ramón Eritja4, Masad Damha5, and Carlos González1
1
Instituto de Química Física Rocasolano, CSIC, 28006 Madrid, Spain.
E-mail: nerea.m@ntu.edu.sg
2
Spectroscopy and NMR Unit, Structural and Computational Biology Programme, CNIO, 28029 Madrid.
3
Joint IRB-BSC program on Computational Biology. IRB-UB, 08028 Barcelona, Spain
4
Institute for Research in Biomedicine, IQAC-CSIC, 08028 Barcelona, Spain
5
Department of Chemistry,McGill University, Montreal, QC, H3A 0B8, Canada
Advances in synthetic chemistry afford new nucleic acids with intriguing properties.
We will present some of our studies on sugar-modified nucleic acids. In particular,
nucleic acids analogs containing 2’fluoro modifications as fluoro-arabino (2’F-ANA),
2’-fluoro-ribose (2’F-RNA) and 2’2’-difluoro-deoxycitosine (2’2’diFC) are
interesting compounds for their potential applications in antigene therapies. In
addition, arabino nucleic acids (ANA) have become attractive systems to construct
genetic systems based on alternative chemical platforms. The preferential
conformations of these analogs are different: ANA, 2’F-ANA and 2’2’diFC are
considered to be DNA analogs, while 2’F-RNA is considered as an RNA-like
nucleotide. By changing the pattern of incorporation of these analogs in a particular
oligonucleotide sequence, structure and stability, as well as binding affinity for RNA
targets can be tuned.
We briefly discuss the structure of several chimeric and hybrid duplexes, whose
sequences combine different patterns of ANA, 2’F-ANA, 2’F-RNA and 2’2’-diFC
nucleotides, as determined by combining 1H and 19F NMR spectroscopy1-3.
The effect of fluorine substitutions on sugars is not only limited to double stranded
structures. Their effects on the structure and stability of guanine quadruplexes,
considered as potential molecular targets, are also very interesting. In bimolecular
human telomeric sequences, 2’F-ANA and ANA modifications are stabilizing while
2’F-RNA destabilizing effect is dramatic. Whereas the native sequence can adopt
different conformations depending on the experimental conditions, a single 2’F-ANA
or ANA substitution provokes the formation of a more stable parallel propeller
structure. Surprisingly, 2’F-RNA substitution disrupt G-quadrplex formation. We
discuss here the three-dimensional solution structure of a 2’F-ANA substituted
telomeric quadruplex 4.
[1] J. K.Watts, N. Martín-Pintado, I. Gómez-Pinto, J. Schwartzentruber, G. Portella, M. Orozco, C.
González and M. J.Damha, Differential stability of 2’F-ANA·RNA and ANA·RNA hybrid duplexes:
roles of structure, pseudohydrogen bonding, hydration, ion uptake and flexibility, (2010) Nucleic
Acids Res. 38, 2498-2511
[2] N. Martín-Pintado, M. Yahayaee-Anzahaee, R. Campos-Olivas, A. M. Noronha, C. Wilds, M. J.Damha
and C. González, The solution Structure of Double Helical Arabino Nucleic Acids (ANA and 2´FANA): effect of arabinose in duplex-hairpin interconversion, (2012) Nucleic Acids Res. 40, 93299340.
[3] N. Martín-Pintado, G. Deleavey, G. Portella, R. Campos-Olivas, M. Orozco, M. J.Damha and C.
González, Stabilizing FC-H…O electrostatic interactions in 2’F-substituted Nucleic Acids. (2013)
Ang. Chem. Int. Ed. Engl. 125, 12287-12290.
[4] N. Martín-Pintado, M. Yahayaee-Anzahaee, G. Deleavey, G. Portella, M. Orozco, M. J.Damha and C.
González, Dramatic Effect of Furanose C2´ Substitutions on Structure and Stability: Directing folding
of the Human Telomeric Quadruplex with a Single Fluorine Atom, (2013) J. Am. Soc. Chem. 135,
5344.
INVITED LECTURES
S8: Membrane biophysics: organization and dynamics.
Chairperson: Francisco Gavilanes (UCM, Madrid)
IL8.1. C2 domains: many ways to recognize specific lipids at the membrane.
M. Senena Corbalán-García (UM, Murcia)
IL8.2. Molecular interactions in living cell membranes, using fluorescence microspectroscopy methods.
Pilar Lillo (IQFR, CSIC, Madrid)
IL8.3. Mechanisms of action of cytotoxic and cytoprotective bile acids – Shaping
membrane structure or permeability?
Manuel Prieto (IST-UTL, Lisboa, Portugal)
IL8.4. Role of curvature in membrane organization.
Vadim A. Frolov (CSIC- UPV/EHU, Bilbao)
C2 DOMAINS: MANY WAYS TO RECOGNIZE SPECIFIC LIPIDS
AT THE MEMBRANE
Senena Corbalan-Garcia and Juan C. Gómez-Fernández
Dpt. of Biochemistry and Molecular Biology A, University of Murcia, Murcia, Spain
senena@um.es
C2 domains are membrane-binding modules that share a common overall fold: a single
compact greek-key motif organized as an eight-stranded anti-parallel β-sandwich
consisting of a pair of four-stranded β-sheets. A myriad of studies have demonstrated that
in spite of sharing the common structural β-sandwich core, slight variations in the residues
located in the interconnecting loops confer C2 domains with functional abilities to
respond to different Ca2+ concentrations and lipids. Our work demonstrates the existence
of a new motif in C2 domains that specifically interacts with phosphoinositides. We
provide structural insights for the C2 domains of PKCα, rabphilin 3A and synatotagmin
1 to explain the ability of different C2 domains to interact with Ca2+ and PI(4,5)P2 and
demonstrate the existence of a specific PI(4,5)P2-binding motif, that confers these
domains with specific properties to dock at the membrane [1, 2]. We have also found that
the C2A domain of synaptotagmin 1 lacks one of these critical lysines, converting the
domain in a non-PI(4,5)P2 responder. Comparison of the affinities these domains exhibit
to interact with membrane models of different phosphoinositides composition suggests
that their differential affinities are controlled by additional amino acidic residues
surrounding the key interacting lysines [2].
These findings provide structural and functional explanation about how these domains
are regulated by a dual-target mechanism and reveal how this family of proteins can
employ subtle structural changes to modulate their sensitivity and specificity to various
cellular signals [3].
Acknowledgements: This work has been sponsored by grants BFU2011-22828 (MINECO, SpainFEDER) and 08700/PI/08 (Fundación Seneca, Region de Murcia).
References
[1] Guerrero-Valero M, Ferrer-Orta C, Querol-Audí J, Marin-Vicente C, Fita I, Gómez-Fernández JC,
Verdaguer N, Corbalán-García S. “Structural and mechanistic insights into the association of
PKCalpha-C2 domain to PtdIns(4,5)P2”. Proc Natl Acad Sci U S A. 106(16):6603-7, 2009.
[2] Guillén J, Ferrer-Orta C, Buxaderas M, Pérez-Sánchez D, Guerrero-Valero M, Luengo-Gil G, Pous J,
Guerra P, Gómez-Fernández JC, Verdaguer N, Corbalán-García S. “Structural insights into the Ca2+
and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1”. Proc Natl
Acad Sci U S A. 110(51):20503-8, 2013.
[3] Corbalan-Garcia S, Gómez-Fernández JC. “Signaling through C2 domains: More than one lipid target”.
Biochim Biophys Acta. 1838(6):1536-1547, 2014.
MOLECULAR INTERACTIONS IN LIVING CELL MEMBRANES
USING FLUORESCENCE MICRO-SPECTROSCOPY METHODS
Carolina García1, Miguel Angel Sacristán1, Alejandro Losada2, José Manuel Molina-Guijarro2,
Carlos M. Galmarini2 and M. Pilar Lillo1
1
Fluorescence and Biophysics Group, Instituto Química Física “Rocasolano”. CSIC. Madrid. Spain.
pilar.lillo@iqfr.csic.es
2
Cell Biology Department, PharmaMar S.A., Colmenar Viejo, Madrid, Spain
Irvalec® and Aplidin® (PharmaMar) are antitumour agents of marine origin. In vitro
treatment of tumor cells with Irvalec® induces necrotic cell death, a process associated
with rapid loss of membrane integrity and subsequent cell permeabilization. Aplidin® is
currently in Phase III trials for multiple myeloma, and its mechanism of action includes
interaction with eEF1A, rapid oxidative imbalance in tumour cells and activation of
signalling pathways leading to cell death apoptosis.
In this work, we have characterized the role of the cell plasma membrane in the
mechanism of action of these compounds [1-2]. With this aim, we have applied state-ofthe-art theoretical and experimental methods, based on picosecond-resolved fluorescence
polarization spectroscopy and two-photon laser excitation microscopy, to provide the
required spatial resolution (Fig. 1). The spectroscopic analysis of various parameters of
the fluorescent bioactive compounds provides valuable information on many chemical
and physical properties of the systems and cellular components.
Figure 1. Identification of different molecular species of Aplidin ® interacting with living cells, using the
FLIM-phasor approach [3]. At short times, Orange species is located in the plasma membrane. Green,
Light Blue and Dark Blue represent the same molecular species, indicating increasing concentrations
with time.
Acknowledgements: This work has been sponsored by CTQ2010-16457, CIBERobn, PharmaMarConsorcio Oncológica (CNIT-E, CDTI) and PharmaMar Contracts. We thank Prof. C. Royer for the
femtosecond laser loan.
References
[1] Molina-Guijarro JM et al., “Irvalec Inserts into the Plasma Membrane Causing Rapid Loss of Integrity
and Necrotic Cell Death in Tumor Cells” PLoS ONE 6-e19042, 2011.
[2] Lillo MP et al., “Irvalec self-organize in the plasma membrane causing rapid loss of integrity and
necrotic cell death in tumor cells” in Proceedings of International Bunsen Discussion Meeting – FRET
in Life Sciences. Gottingen, 2011.
[3] Digman, M. and Gratton E., “Fluorescence Lifetime Microscopy: The Phasor Approach”,
Comprehensive Biophysics 2, 24-38, 2012.
MECHANISMS OF ACTION OF CYTOTOXIC AND CYTOPROTECTIVE
BILE ACIDS – SHAPING MEMBRANE STRUCTURE OR PERMEABILITY?
Tânia Sousa1, Ana Coutinho1,2, Rui E. Castro3, Sandra Pinto1, Susana D. Lucas3, Rui Moreira3,
Cecília M.P. Rodrigues3, Fábio Fernandes1 and Manuel Prieto1
1
Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology, Instituto
Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
manuel.prieto@tecnico.ulisboa.pt
2
Dep. Química e Bioquímica, FCUL, Campo Grande, 1749-016 Lisboa, Portugal
3
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy,
University of Lisbon, Lisbon, Portugal
Deoxycholic acid (DCA) is an apoptotic bile acid at submillimolar concentrations. On the
other hand, ursodeoxycholic acid (UDCA) prevents apoptosis in the same concentration
range. The mechanisms that trigger these opposite signaling effects are still unclear. We
have recently shown that these bile acids exhibit low partition to cholesterol-rich (liquid
ordered,lo) membranes and that DCA and other apoptotic bile acids are able to partially
disrupt the ordering of lipid model membranes by cholesterol in liquid disordered
membranes (ld) [1].
Using fluorescence microscopy methodologies, we show that fluorescent derivatives of
DCA and UDCA are present at very low concentrations in the plasma membrane of both
HEK293 and hepatocyte living cells, possibly as a consequence of low partition of bile
acids to cholesterol-rich membranes. Additionally, both apoptotic and cytoprotective
unlabeled bile acids have no effect on the fluidity of the plasma membrane at apoptotic
concentrations. However, fluorescent derivatives of bile acids are found significantly
enriched in the mitochondrial membrane of hepatocytes. These results suggest that the
modulation of apoptosis by bile acids is not the result of modulation of plasma membrane
structure and are likely associated with mitochondria damage/protection.
Acknowledgements: This work was supported by FCT-Foundation of Science and Technology
(PTDC/QUI-BIQ/119494/2010, RECI/CTM-POL/0342/2012). T.S. and F.F. acknowledge research
grants (SFRH/BD/92398/2013 and SFRH/BPD/64320/2009) from FCT.
Reference
[1] J. Mello-Vieira et al. Cytotoxic bile acids, but not cytoprotective species, inhibit the ordering effect of
cholesterol in model membranes at physiologically active concentrations. Biochim. Biophys. Acta –
Biomembranes, 1828, 152-63, 2013.
ROLE OF CURVATURE IN MEMBRANE ORGANIZATION
Vadim Frolov1,2, Artur Escalada1, Pavel Bashkirov3, Peter Kuzmin3 and Anna Shnyrova1
1
Biophysics Unit (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology,
University of the Basque Country, Leioa, Spain
vadim.frolov@ehu.es
2
IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
3
A.N. Frumkin Institute of Physical Chemistry and Electrochemistry,
Russian Academy of Sciences, Moscow, Russia
Membrane curvature is an important factor in lateral organization of cellular membranes.
Curvature sensing by individual membrane proteins and lipids has been explored in
details. Much less studied is the collective behavior of membrane components in
curvature gradients. Here we demonstrate that curvature-driven sorting of core membrane
components affects the apparent elasticity of membrane domains. Redistribution of lipids
substantially diminished bending rigidity thus facilitating membrane remodeling at high
curvatures. Curvature-driven condensation of proteins, in turn, stabilizes low-curved
membrane domains. The implications of these findings for regulation of vesicular
transport in cells are discussed.
Acknowledgements: this work has been sponsored by Spanish Ministry of Economy and
Competitiveness, grant BFU2012-34885, and Basque Government, grant IT838-13.
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José María
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Barcelona Supercomputing Center
Southern Denmark University
King's College London
IQFR, CSIC
IST - University of Lisbon
Institute of Biomedicine of Valencia, CSIC
CIB, CSIC
University of Valencia
University of Extremadura
University of Zaragoza
Pharmamar
Center for Genomic Regulation
Universidade Nova de Lisboa
University of Lisbon
Paul Scherrer Institute
University Miguel Hernández
CIC BioGUNE
Valencia
Barcelona
Odense
Krakow
London
Madrid
Barcelona
Madrid
Murcia
Barcelona
Lisbon
Madrid
Madrid
Madrid
Leioa
Madrid
Valencia
Madrid
Granada
Madrid
Burjassot
Badajoz
Alcalá de Henares
Valencia
Zaragoza
Madrid
Barcelona
Caparica
Lisbon
Villigen
Elche
Bilbao
Madrid
Madrid
Spain
Spain
Denmark
Poland
United
Spain
Spain
Spain
Spain
Spain
Spain
Portugal
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Spain
Portugal
Portugal
Switzerlan
Spain
Spain
Spain
Spain
morzaez@cipf.es
chiara.pallara@bsc.es
parra@sdu.dk
marta.pasenkiewicz-gierula@uj.edu.pl
judit.perales-calvo@kcl.ac.uk
jmperez@iqfr.csic.es
m15perezver@hotmail.com
jesusperezgil@gmail.com
mps66975@um.es
s.poly@nanogune.eu
mpons@ub.edu
manuel.prieto@tecnico.ulisboa.pt
erney@cib.csic.es
areygayo@ucm.es
grivas@cib.csic.es
eva.rodriguez.hortelano@gmail.com
javierr@cib.csic.es
rubio@ibv.csic.es
laura_ruiz@cib.csic.es
druzafa@gmail.com
gonsaez@cib.csic.es
jesus.salgado@uv.es
aksamhan@unex.es
pedroalejandro.sanch@uah.es
msancho@cipf.es
jsancho@unizar.es
gsnunez@pharmamar.com
luis.serrano@crg.es
t.simoes@campus.fct.unl.pt
gsoveral@ff.ul.pt
michel.steinmetz@psi.ch
ftaberner@umh.es
mvalle@cicbiogune.es
jmv@cnb.csic.es
soniavaz@ucm.es
Verdaguer Massana
Verdiá Báguena
Vergoñós Tomàs
Vilar Cerveró
Vögler
Wypijewska del Nogal
Zamora Carreras
Zorrilla López
Nuria
Carmina
Albert
Marcial
Bernhard Oliver
Anna
Héctor
Silvia
Molecular Biology Institute of Barcelona
University Jaume I
CIB, CSIC
Institute of Health Carlos III
University of Warsaw
IQFR, CSIC
Barcelona
Castellón de la Plana
Madrid
Majadahonda
Palma de Mallorca
Warsaw
Madrid
Madrid
Spain
Spain
Spain
Spain
Spain
Poland
Spain
Spain
nvmcri@ibmb.csic.es
verdia@uji.es
avergonyos@cib.csic.es
mvilar@isciii.es
oliver.vogler@uib.es
wypijewska.del.nogal@gmail.com
hzamora@iqfr.csic.es
silvia@cib.csic.es

ORAL COMMUNICATIONS S1: Supramolecular Assemblies

Transcription

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