Characterization of a conformationally sensitive TOAC spin

peptides 29 (2008) 1919–1929
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Characterization of a conformationally sensitive TOAC
spin-labeled substance P
Aaron M. Shafer a, Clovis R. Nakaie c, Xavier Deupi d, Vicki J. Bennett b, John C. Voss a,*
a
Department of Biochemistry & Molecular Medicine, University of California, Davis, CA 95616, United States
Department of Neurobiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272, United States
c
Departamento de Biofisica Universidade Federal de São Paulo Escola Paulista de Medicina, Brazil
d
Laboratory of Computational Medicine, Biostatistics Unit, School of Medicine, Universitat Autonoma de Barcelona, 08193 Bellaterra,
Barcelona (Catalunya), Spain
b
article info
abstract
Article history:
To probe the binding of a peptide agonist to a G-protein coupled receptor in native
Received 9 May 2008
membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethyl-
Received in revised form
piperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P
31 July 2008
peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. The affinity of
Accepted 1 August 2008
the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC
Published on line 15 August 2008
derivative is 250-fold lower. Both peptides activate receptor signaling, though the potency
of the 9-TOAC peptide is substantially lower. The utility of these modified ligands for
Keywords:
reporting conformational dynamics during the neurokinin-1 receptor activation was
Substance P
explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC
GPCR
nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC
TOAC spin label
substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is
EPR
detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that
ESR
maintain the receptor in its high-affinity binding state. In contrast, 4-TOAC substance P
binding is observed by solution EPR under both low- and high-affinity receptor states, with
evidence of a more strongly immobilized peptide in the presence of GDP. In addition, to
better understand the conformational consequences of TOAC substitution into substance P
as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC
versions of the peptide were constructed, and the molecular dynamics calculated via
simulated annealing to explore the influence of the TOAC substitutions on backbone
structure.
# 2008 Elsevier Inc. All rights reserved.
* Corresponding author. Tel.: +1 530 754 7583; fax: +1 530 752 3516.
E-mail address: jcvoss@ucdavis.edu (J.C. Voss).
Abbreviations: NK1r, neurokinin-1 receptor; SP, substance P; 4-TOAC SP, TOAC substance P with TOAC integrated at position 4; 9-TOAC
SP, TOAC substance P with the TOAC spin label integrated at position 9; Nk1r, neurokinin-1 receptor; EPR, electron paramagnetic
resonance spectroscopy; CHO, chinese hamster ovary cells; GFP, green fluorescent protein; GPCR, G-protein coupled receptor; Gpp(NH)p,
guanylyl-imidodiphosphatetetralithium salt; TFA, trifluoroacetic acid; BCA, bicinchoninic Acid; TOAC, 2,2,6,6-tetramethylpiperidine-Noxyl-4-amino-4-carboxylic acid; BSA, bovine serum albumin; MBB, membrane binding buffer; BBA, binding buffer A; FBS, fetal bovine
serum; TBS, Tris-buffered saline; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high performance liquid chromatography.
0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2008.08.002
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1.
peptides 29 (2008) 1919–1929
Introduction
G-protein coupled receptors (GPCRs or 7 TM receptors)
represent a superfamily of cell surface integral membrane
proteins characterized by having a common template composed by seven membrane-spanning alpha helices joined by
hydrophilic loops. These receptors are capable of binding a
wide of array of chemically and structurally diverse extracellular ligands. Binding results in the generation of an
intracellular signal, through the activation of cytoplasmic G
proteins [4,20]. Numerous radioligand binding studies suggest
that, in the absence of ligand, GPCRs exist in equilibrium
between a low affinity state, where the G-protein is not
coupled to the receptor, and a high-affinity state, where the
receptor is pre-coupled to the G-protein [25]. In addition,
recent work in the b2 adernergic receptor and the neurokinin 2
receptor [37,51], show that GPCRs can adopt multiple ligandspecific conformations [14,25,26]. This feature provides a given
GPCR with the capability to signal distinct pathways according
to the ligand-specified conformational state.
In this study the interaction between Substance P and the
Tachykinin 1 receptor (Nk1r)1 was investigated. The receptor,
also designated the substance P or neurokinin-1 receptor,
has been implicated in numerous physiological functions,
which include modulatory roles in the central nervous
system and proinflammatory action (for review see [30]).
Although much debate still exists as to which neurokinin
represents the physiological ligand for the receptor, the
substance P neurokinin has been identified as the primary
ligand [31].
The Nk1r receptor-bound conformation of SP has been
inferred from a series of spectroscopic structural studies [38],
and from NMR and modeling studies [41,44]. In summary, it
has been proposed that SP has a highly flexible N-terminal
domain (residues Arg1-Pro2-Lys3) projecting toward TM1 and
TM2 and exposed to the extracellular loops and/or to the
solvent. The central part of the ligand (residues Pro4-Gln5Gln6-Phe7-Phe8) forms a core helical structure and establishes
important interactions with EC2 and EC3. This helical core
structure has been observed in methanol and in micellar
media, and has been validated as a bioactive conformation by
the design of highly potent cyclic analogs of substance P that
mimic this structure [40]. The C-terminus of SP (residues Gly9Leu10-Met11) is located in proximity to TM5 and TM6, deeper
into the central core of the receptor, adopting preferentially a
conformation similar to a polyproline II helix or a beta strand.
Peptide backbone flexibility is essential to precisely position
the crucial recognition side chains, as positions 7, 10, and 11. In
addition, position 9 in SP has been found to constitute a hinge
point for recognition discrimination between two binding sites
[44].
Biophysical methods that utilize site-specific protein
labeling are suitable for detecting conformational states of
the ternary complex of receptor, ligand and G-protein
[21,50,51,58]. However, these approaches are hampered due
to the difficulty in over-expressing, purifying and reconstituting the receptor and the G-protein. To overcome these
difficulties, the ligand can be used as a probe to investigate
G-protein dependent receptor conformational states. This
approach has been particularly useful for investigating ligand–
receptor interactions in peptide hormone GPCRs [36,53,54],
such as the Nk1r, which has a nanomolar affinity for the
substance P (SP) peptide. For example, the SP binding pocket in
Nk1r was probed using a SP peptide labeled at Lys3 with
fluorescein [53], or danysyl fluorophores [56]. In the former
case, a decrease in the attached fluorescein anisotropy occurs
in the presence of the hydrolysis-resistant GTP analogue
GMPPNP. However, these measurements were complicated by
the difficulty in resolving the increased mobility in bound
peptide from the increased level of dissociated peptide [53].
To address these problems, we have previously attached a
nitroxide spin label to Lys3 of SP peptide for examination by
electron paramagnetic (EPR) spectroscopy [49]. However, the
high flexibility of the spin-labeled lysine made this probe less
than ideal for detecting alternate affinity states associated
with G-protein coupling [49]. Therefore, to reduce the noise
induced by side chain flexibility, we sought to attach a spin
probe at a location more closely associated with the SP
peptide backbone. For this purpose, we have synthesized SP
peptides containing the rigid nitroxide side chain TOAC
(Scheme 1) [33]. TOAC has a higher probability of detecting
slight mobility changes, and has been incorporated into other
peptide ligands by solid phase synthesis [3,17,34,35,46]. Here,
we show integration of the TOAC spin label at positions 4 and
9 of SP peptide (4-TOAC SP and 9-TOAC SP, respectively)
results in biologically active peptides. We have also demonstrated that 4-TOAC SP binding to the Nk1r is sufficient for
reporting changes in conformational dynamics. Furthermore, unlike the SP probe containing a spin label attached
to Lys 3, 4-TOAC SP is sensitive to detecting conformational
changes that occur upon changes of the association of the
receptor with G-protein [25].
2.
Materials and methods
2.1.
Materials
Substance P was purchased from Peninsula Laboratories (San
Carlos, CA). Collagenase, ionomycin, bacitracin, leupeptin,
20 g/ml chymostatin were purchased from Sigma. The
protease inhibitor cocktail was from Calbiochem. Fluo-3-AM
was from Molecular probes and pluronic F-127 was from Texas
Fluorescence Labs. 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylate N-hydroxysuccinimide ester was from Toronto
Scheme 1 – Structure of 2,2,6,6-tetramethylpiperidine-1oxyl-4-amino-4-carboxylic acid (TOAC).
peptides 29 (2008) 1919–1929
Research Chemicals. GTPgS was obtained from Biomol. (3H)-SP
was purchased from Amersham.
2.2.
Synthesis of TOAC-labeled Substance P
2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic
acid (Scheme 1) was incorporated at positions 4 and 9 (4-TOAC
SP or 9-TOAC SP) of the substance P sequence according to a
previous strategy [32,35]. In this method the 9-fluorenylmethyloxycarbonyl (Fmoc) and tert-butyl (t-But) strategies [2,18], were
combined for peptide chain assembly and final cleavage from
the resin. The two paramagnetic peptides were synthesized in
methylbenzhydrylamine-resin (0.3 mmol/g) using the following Fmoc-amino acid protecting groups: 2,2,5,7,8-pentamethylchroma-6-sulfonyl (Pmc) for Arg and t-But for Lys. Couplings
were carried out with a 1:1:1 mixture of Fmoc-amino acid/O-(7azabenzotriazol-1-yl)-1,13,3-tetramethyluronium-hexafluorophosphate (HATU)/1-hydroxy-7-azabenzotriazole (HOAt) in the
presence of N,N-diisopropylethylamine in 1-methyl-2-pyrrolidinone (NMP). Due to the very high difficulty in coupling the
next amino acid to the TOAC residue, more severe acylation
conditions were applied (fivefold excess of reagents and
repeating the coupling step twice).
The peptides were cleaved from the resin with anhydrous
solution
of
HF:o-cresol:dimethylsulfide:ethanedithiol
(9.0:0.5:0.5, v/v) at 0 8C for 2 h. After evaporation, the resin
was washed with ethyl acetate, dried and the peptides were
extracted into 5% acetic acid and lyophilized. The crude TOACattaching peptides were submitted to alkaline treatment (pH
10, 1 h, 50 8C) for complete reversal of the nitroxide protonation that occurs during the final acid cleavage in HF. The
peptides were purified by HPLC, using a C18-column and
aqueous 0.02 M ammonium acetate (pH 5) and 60% acetonitrile
solutions as solvent A and B, respectively (linear gradient of
30–70% B in 2 h). Fractions that contained >97% were
combined, lyophilized and yielded 12 and 8 mg 9-TOAC and
4-TOAC substance P, respectively. The homogeneity of both
purified peptides was checked by amino acid analysis,
analytical HPLC and electrospray liquid mass spectrometry.
4-TOAC substance P: m/z = 1,447.79 [M+H+]; obtained: 1447.90
and 9-TOAC substance P: m/z = 1,487.85 [M+H+]; obtained
1487.60.
2.3.
Growth and transfection of CHO cells expressing the
Nk1r-GFP fusion protein
The rNK1GFP cDNA was stably transfected into CHO cells
using the Flp-IN system (Invitrogen, Carlsbad, CA), as
previously described [49]. The engineered recombination site
in this system, combined with enhanced selection by FACS
based on the GFP fluorescence, provides surface expression
levels of 4 106 receptors/cell. A detailed protocol for the
stable transfection of peptide receptors in CHO cells has been
recently published [48].
2.4.
Membrane isolation
Membranes were prepared from 3.5 107 CHO cells from five
100 mm cell culture plates. The plates were rinsed two times
with phosphate buffered saline. All subsequent steps were
1921
carried out at 4 8C. Cells were harvested by scraping in 15 ml of
a lysis buffer containing 10 mM Tris (pH 7.4) 1 mM EDTA, 10 g/
ml benzamidine and 10 g/ml leupeptin. The cells were then
lysed using a 15 ml tight glass douncer with 100 strokes or until
no intact cells remained as assessed by conventional light
microscopy. The homogenate was centrifuged at 800 g for
10 min. The pellet was discarded and the supernatant was
then centrifuged at 20,000 g for 20 min. The pellet was
resuspended in 1 ml of storage buffer (50 mM Tris–HCl pH 7.4,
3 mM MgCl2 and 100 protease inhibitor) at a protein
concentration of 1–5 mg/ml and stored at 80 8C until further
use.
2.5.
TOAC SP radioligand binding to isolated membranes
The binding affinities for 4-TOAC SP and 9-TOAC SP were
determined by competition assays using tritium labeledsubstance P ([3H]-SP; NEN, Boston, MA). Membrane pellets
were diluted into binding buffer (storage buffer with 0.04 mg/
ml BSA and 100 mM NaCl) to a final protein concentration of
5 mg/ml for each 200 ml binding reaction. Assays were carried
out in a Millipore 96 well 0.22 mm GV-Durapore membrane
plate using a concentration of 0.8 nM [3H]-SP. The TOAC
peptides were added at increasing concentrations, from
100 pM to 1 mM, and incubated for 1 h at room temperature
and then kept at 4 8C until vacuum filtration. Membranes were
then washed with three rinses of 100 ml of ice-cold rinse buffer
(25 mM Tris, pH 7.5 3 mM MgCl2, 1 mM EDTA). Filters were
punched and the remaining (bound) radiolabeled SP measured
by scintillation counting using a Beckman LS 60001. Nonspecific binding was specified as the counts obtained with
10 mM unlabeled substance P in the presence of [3H]-SP.
Specific binding was determined by subtracting nonspecific
binding from the original cpm measurements.
2.6.
TOAC SP activation properties in CHO cells
The ability 4-TOAC SP and 9-TOAC SP to activate the Nk1r was
examined by measuring increases in intracellular Ca2+.
Chinese hamster ovary cells (CHO) stably transfected with
the cDNA of the rat Nk1r were obtained from James E. Krause
(Neurogen Corporation, Branford, CT) and maintained as
previously described [42]. These CHO cells were cultured into
Falcon 96-well plates and loaded with Fluo 3-AM (Molecular
Probes, Eugene, OR), a Ca2+ indicator dye, with 0.1% pluronic F127 (Texas Fluorescence Labs, Austin, TX) for 30 min at 37 8C in
an extracellular solution (ES) containing: 10 mM HEPES, 10 mM
glucose, 115 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.3 mM CaCl2
and 2.5 mM probenecid. Cytosolic Ca2+ concentrations were
measured using a FluoSTAR fluorescent plate reader (BMG
Labtechnologies, Durham, NC) by measuring the fluorescence
emission at 520 nm and excitation at 480 nm.
Dose–response curves were obtained by measuring the
Ca2+ responses produced by adding various concentrations
(1 pM, 100 pM, 1 nM, 10 nM, 100 nM and 1 mM) of SP, 4-TOAC SP
or 9-TOAC SP. Ionomycin (Sigma, St. Louis, MO) was added at a
concentration of 10 mM at the end of each experiment to
normalize between experiments. Ca2+ responses were measured by subtracting the peak of each response from the
baseline. Responses are expressed as a fraction of the
1922
peptides 29 (2008) 1919–1929
ionomycin response. The experiment was performed three
times providing a sample number of at least 10 for each
concentration.
2.7.
EPR measurements of 4-TOAC SP and 9-TOAC SP
For EPR measurements, membranes were isolated from 108
CHO cells expressing the Nk1r-GFP chimera, as described
above for competition binding studies. Membrane pellets were
resuspended in 1 ml of binding buffer at a protein concentration of 3.5 mg/ml. The membrane fractions were then
incubated at 4 8C for 2 h in the presence of 1 mM 4-TOAC SP
or 9-TOAC SP. The fractions were then centrifuged at
20,000 g for 5 min, washed by resuspending in 1 ml of
binding buffer, re-centrifuged and then resuspended in
binding buffer to a protein concentration of 70 mg/ml. For
each sample, 5 ml of membrane suspension was then loaded
into a glass capillary and analyzed as described above for the
live cells but at room temperature. Nonspecific binding was
examined using membrane preparations isolated from CHO
cells lacking the Nk1 receptor. To access nucleotide effects,
membrane-peptide incubations were also made in the binding
buffer containing either 200 mM GTPgS or 1 mM GDP, levels
sufficient to saturate the endogenous G proteins [49]. The
respective nucleotide levels were also included in the binding
buffers used for the wash and resuspension steps.
EPR measurements were performed using a JEOL X-band
spectrophotometer fitted with a loop gap resonator (Molecular
Specialties, Inc., Milwaukee, WI). All scans were performed at
room temperature (22–25 8C). Spectra were obtained by signal
averaging three scans over 100 G at a microwave power of
2 mW, a time constant of 0.3 s, a scan time of 2 min and a
modulation amplitude of 1 G.
2.8.
Computational methods
In order to predict the structural dynamic consequences of
TOAC substitution into the SP peptide, we built a computational
model of this peptide as follows. A FASTA (version 34.26 January
12, 2007) search for sequence similarity of the SP sequence
against the PDB Protein Structure Database library as implemented in the EBI web site resulted in two hits, the tachykinin
peptides uperolein and kassinin. We used kassinin (Swiss Prot
accession number: P08611; PDB id: 1MYU) as it possesses the
highest sequence identity (70%) with substance P. The structure
of this peptide has been resolved by NMR in both aqueous
environment and in dodecylphosphocholine (DPC) micelles.
The 20 NMR-derived structures of kassinin where used to obtain
a first three-dimensional model of substance P using Modeler
[45]. Subsequently, the backbone dihedral angles of the residues
Leu10 and Met11 where changed according to the values
proposed by Sagan et al. [44], which correspond to the
conformation of the C-terminal required for binding and
activation of the neurokinin-1 receptor. These values are
phi(Leu10) 608; psi(Leu10) 1508 and phi(Met11) 608, which
correspond to an extended conformation close to a polyproline
II structure. Psi(Met11) was set to 1508, corresponding to the
polyproline II value. The structure of this initial model was
optimized with 500 steps of energy minimization. In order to
explore the conformational space available to the residues Pro-4
and Gly-9 in this model of substance P, two independent 500 ps
molecular dynamics simulations with annealing (heating to
400 K in 20 ps, maintaining this temperature during 150 ps,
cooling down to 300 K in 30 ps and letting run for 300 ps) were
run on the model peptide. The backbone dihedrals of the rest of
the peptide (residues 1–3, 5–11 and 1–8, 10–11, respectively) were
restrained to their initial positions; the restraint is a well with a
square bottom (28 from the initial value) with parabolic sides
out to a defined distance (38 from the initial value), and then
linear sides beyond that, with a force constant for the restraint
energy of 200 kcal/mol. Molecular mechanics parameters for
the spin label TOAC were obtained using the program
ANTECHAMBER, using the general amber force filed, and
atomic charges derived using the restrained electrostatic
potential (RESP) method on a electrostatic potential calculated
ab initio with the HF/6-31G* basis set. An extra improper torsion
was added to keep the nitroxide group planar, using the force
value of Improta and Barone [24]. The model of substance P was
labeled separately in silico at positions 4 and 9 with TOAC (Fig. 4),
and molecular dynamics simulations with an identical setup
were run for these two systems. The simulations were carried
out with the Sander module of AMBER 9 (AMBER 9, University of
California, San Francisco) using the ff03 [15], force field, SHAKE
bond constraints in all bonds, a 2-fs integration time step, at
constant volume and using the particle mesh Ewald method to
compute electrostatic interactions [11].
3.
Results
3.1.
Pharmacology of the spin-labeled substance P
To determine the effect of the TOAC substitutions on the
functional properties of SP, the binding of the modified
peptides was measured using membranes isolated from the
CHO cell-line stably expressing the Nk1-GFP protein. Fig. 1
Fig. 1 – Effect of the TOAC spin label substitutions on the
binding affinity for substance P. Competition assays were
carried out on CHO cell membranes expressing the Nk1rGFP. The fraction of [3H]-SP bound to membranes was
measured as a function of 4-TOAC SP (&), 9-TOAC SP (~),
or the non-labeled ligand (*). All assays were performed
in triplicate in three separate experiments. Error bars
represent the standard error of the mean from two
independent experiments giving a total of n = 6
measurements.
peptides 29 (2008) 1919–1929
1923
receptor show a signal from the spin-labeled peptide. To avoid
complications from a slow dissociation of peptide during the
experiment, signal averaging was not performed, and only a
single 2 min scan was acquired for each sample. The spectrum
of 4-TOAC SP bound to membranes isolated from CHO cells
over-expressing the Nk1r-GFP protein is shown in Fig. 3A (black
trace), corresponding to a moderately immobilized peptide.
Addition of an excess of native SP peptide to the sample
Fig. 2 – Effect of TOAC-modified SP peptides on Nk1r
activation. The activating properties as measured by
intracellular calcium mobilization were measured as a
function of agonist concentration. Both the 4-TOAC SP (&)
and 9-TOAC SP (~) spin-labeled analogues retain activity,
however the potency of the 9-TOAC SP is significantly
reduced compared to the non-labeled agonist (*). All
assays were performed in triplicate in three separate
experiments. Errors represent the standard error of the
mean for n = 10 measurements.
shows binding analysis for 4-TOAC SP and 9-TOAC SP, as
measured by competition against a radiolabeled native SP
agonist. The IC50 value for 4-TOAC SP is nearly identical to
native SP, giving values of 1.7 and 1.6 nM, respectively.
However, substitution at position 9 with TOAC leads to a
large decrease in binding affinity, with a measured IC50 value
of 488 nM for this peptide.
To further characterize the functional properties of 4-TOAC
SP and 9-TOAC SP, we measured the ability of the peptides to
activate signaling by measuring Nk1r-dependent calcium
mobilization in CHO cells expressing the receptor. The EC50
for the labeled peptides were measured in CHO cells expressing
the Nk1r, where the amplitude of receptor activation can be
readily observed through the mobilization of internal calcium
stores [29]. As shown in the dose–response curves of Fig. 2, 4TOAC SP invoked a more potent response than SP, with a 10-fold
increase (relative to native SP), as judged by the EC50. The
substitution of TOAC at position 9 however leads to a less potent
agonist, as indicated by a 20-fold decrease (relative to native SP)
in the EC50. The EC50 values for 4-TOAC SP and 9-TOAC SP and SP
were 0.58, 117.61 and 5.80 nM, respectively. In contrast, no
response is detected with either 4-TOAC SP, 9-TOAC SP or SP in
non-transfected cells (not shown).
3.2.
Binding of 4-TOAC SP and 9-TOAC SP as
observed by EPR
The dynamics of the TOAC-substituted SP peptides was
evaluated by EPR spectroscopy of the peptides bound to
membranes isolated from CHO cells expressing the Nk1r-GFP
protein. After incubation and removal of unbound peptide, only
the membranes derived from the CHO cells over-expressing the
Fig. 3 – EPR spectra of 4-TOAC SP and 9-TOAC SP bound to
membranes from CHO cells expressing the Nk1 receptor.
(A) Binding of 4-TOAC SP to membranes containing Nk1rGFP results in a moderately immobilized spectrum (black
trace). Upon addition of excess native SP, the spectrum
becomes narrower (red trace), identical to the free peptide
in solution. (B) The more immobilized spectrum of 9-TOAC
SP is only detected when the incubation and wash
treatments contain GDP. (C) Effect of nucleotides on the
EPR spectra of 4-TOAC SP with membranes from CHO cells
expressing Nk1r-GFP. Binding efficiency of 4-TOAC SP is
increased in the presence of GDP (black trace) compared to
GTPgS (red trace). In addition, the presence of GDP
produces a prominent population of more immobilized
spins (arrows) Spectrometer gain and NK1r levels are
equivalent for all spectra. Intensities vary according to the
total amount bound and the line widths of the spectral
components. (For interpretation of the references to color
in this figure legend, the reader is referred to the web
version of the article.)
1924
peptides 29 (2008) 1919–1929
containing bound 4-TOAC SP results in a sharp isotropic signal
(identical to the free peptide), demonstrating a chase of 4-TOAC
SP off the receptor (Fig. 3A, red trace). Due to its lower binding
affinity, the EPR spectrum of the 9-TOAC SP peptide incubated
with the same amount of membranes was only observed in the
presence of 1 mM GDP (Fig. 3B), consistent with this treatment
inducing a higher affinity binding of ligand (see below). In the
presence of GDP, the resulting spectrum of membranes
incubated with 9-TOAC SP is much broader than is obtained
with 4-TOAC SP, consistent with earlier notions that this region
of the agonist resides within a hydrophobic binding pocket in
the central core of the receptor, near to TM5 and TM6 [1,12,38]. It
should also be noted that due to its lower binding affinity, the
spectrum obtained with the 9-TOAC SP peptide (Fig. 3B)
contains a sharp component representing free peptide. The
amplitude of this highly mobile component increases with time
(3% per minute; not shown), consistent with a slow dissociation of the peptide off the receptor.
The Nk1r is known to display high and low affinity ligand
binding states dependent on its association with a G-protein
[8,49]. To test whether the TOAC SP ligands can report receptor
coupling by endogenous G protein, isolated membranes from
CHO cells expressing the Nk1r-GFP protein were homogenized
in the presence of GDP or the hydrolysis-resistant GTP analogue
GTPgS. In the presence of GDP, the resulting spectrum of bound
4-TOAC SP is of significantly greater intensity than the signal
obtained in the presence of GTPgS (Fig. 3C). This indicates that
less peptide is washed off in the presence of GDP than with
GTPgS, where the incubations contained equal amounts of CHO
membranes and 4-TOAC SP. In addition, a broader component is
more apparent in the spectrum of bound 4-TOAC peptide
(Fig. 3C, arrows) when GDP is included in the treatment, though
our ability to clearly resolve the hyperfine extrema is limited due
to the low signal:noise ratio. However, this result suggests that a
large fraction of the bound 4-TOAC peptide experiences a
substantial loss in motional freedom in the presence of GDP,
when compared to the magnitude of the immobilized component following GTPgS treatment. Again, due to the lower binding
affinity of Nk1r for 9-TOAC SP, an appreciable signal for bound
peptide was only obtained in the presence of GDP (Fig. 3B).
Therefore, 9-TOAC samples with GTPgS or no nucleotide
addition could not be analyzed for line shape properties (Fig. 4).
3.3.
Molecular dynamics of model peptides
In order to gain insight into the structural effects of the TOAC
substitutions on SP we performed a series of molecular
dynamics simulations (see Section 2) on the SP peptide and its
labeled analogs 4-TOAC SP and 9-TOAC SP. Fig. 5 shows the
distribution of the backbone dihedral angles f and w of position
4 (left panels) and 9 (right panels) of SP (top panels) and TOAClabeled SP (bottom panels) during the simulations. For
unlabeled SP, f and w of residue Pro-4 (top-left panel) are
clustered in the a-helical region, i.e. Pro-4 maintains the initial
a-helical conformation during the whole trajectory. However,
f and w of residue Gly-9 (top-right panel) depart from their
roughly a-helical initial values (red circle) to a structure
resembling to a left-handed helix once the structure gets
equilibrated. For the 4-TOAC SP labeled peptide, the distribution of f and w of the TOAC label at position 4 (bottom-left
Fig. 4 – Computational model of substance P (see Section 2).
The address sequence (residues 1–3), responsible for
receptor specificity, is highlighted in orange, and the
message sequence (residues 7–11), responsible for
activation, is highlighted in green. The TOAC spin labels
introduced separately at positions 4 and 9 are shown as
sticks. The figure shows how 4-TOAC is located exactly in
the interface between the highly flexible address sequence
and the helical core of the peptide, while 9-TOAC is located
in the message sequence, in a position found to constitute
a hinge point for recognition discrimination between two
binding sites (see Section 1). These structural properties
are consistent with the findings described here and
elsewhere: a N-terminal ‘‘address’’ domain remains
highly flexible in the bound state [38,44,49]; a helical core
region (residues 4–8) proposed to interact with EC2 and
EC3 [44]; and a C-terminal ‘‘message’’ domain, predicted to
adopt a polyproline II extended helical conformation that
binds in a pocket comprised of receptor TMs 5 and 6 [41].
This figure has been created with PyMOL (DeLano, W.L.
The PyMOL Molecular Graphics System (2002) on World
Wide Web http://www.pymol.org). (For interpretation of
the references to color in this figure legend, the reader is
referred to the web version of the article.)
panel) is very similar to Pro-4 of the unlabeled peptide
(compare to top-left panel), i.e. the spin label maintains its
initial a-helical conformation just like the Pro residue at the
same position in the unlabeled peptide. Interestingly, introduction of the TOAC spin label at position 9 (i.e. replacing Gly9) leads to an extension of the conformational space available
to this position (see bottom-right panel). While the labeled
peptide stabilizes also in the left-handed helix region, it is also
capable to adopt conformations of a beta-sheet, which were
not available to the unlabeled peptide.
4.
Discussion
In this work we have combined pharmacological data and EPR
spectroscopy to study the value of TOAC-labeled SP as a
reporter of the conformational changes of the neurokinin-1
receptor during its activation. In addition, we have used
peptides 29 (2008) 1919–1929
1925
Fig. 5 – The distribution of the backbone dihedral angles f and w of position 4 (left panels) and 9 (right panels) of SP (top
panels) and TOAC-labeled SP (bottom panels) during the molecular dynamics simulations. Introduction of the TOAC label at
position 4 does not alter the alpha helical geometry of this position (compare top-left to bottom-left panels). However,
labeling at position 9 extends the conformational space of the peptide (compare top-right to bottom-right panels). These
results provide a structural basis for the minimal effect on SP activity with the TOAC substitution at Pro-4, as well as
demonstrating how substitution at Gly-9 results in a peptide with a loss of hinge motion between the binding domains [44].
The panel backgrounds have been adapted from http://www.schematikon.org/.
molecular dynamics simulations to analyze how the structural properties of SP are affected by the introduction of TOAC
spin label.
4.1.
TOAC-labeled SP as a reporter of Nk1r activation
Within the family of tachykinin peptides, the conserved
pentapeptide (Phe-X-Gly-Leu-Met-NH2) in the agonist’s C-
terminal portion (the ‘‘message sequence’’) is responsible for
receptor activation, whereas the divergent N-terminal portion
(the ‘‘address sequence’’) provides specificity for the target
receptor [1,39,47]. Although the sequence of SP (RPKPQQFFGLM)
has been mutated at several locations, it is difficult to predict the
functional consequences of residue replacement at a particular
position. Previous studies using dansyl or fluorescein SP
derivatives showed that substitutions at the N- and C-termini
1926
peptides 29 (2008) 1919–1929
retained activity, while substitution at Phe-8 results in an
agonist with very little activity [55]. However, due to the
heterogeneity of conformations found in solution and lipid
environments, the structural basis of these functional properties in receptor binding and activation remain elusive. The mid
region (residues 4–9) of SP may adopt a helical conformation in
the receptor-bound state, and that Gly9 constitutes a hinge
point in the peptide, placing Leu10 and Met11 into a hydrophobic binding pocket [1,12]. Therefore, we introduced the
TOAC spin label at positions 4 and 9, at the beginning and end of
the helical central region of SP. Since the tetra-substitution at
the Ca imposes a strong structural constraint without disrupting alpha helical structure [52], TOAC can serve as a helix
stabilizer [19]. Then, we tested the ability of the labeled SP to
bind to and activate the receptor.
4.2.
9-TOAC disrupts the structure and pharmacological
profile of SP
It has been proposed that a hinge at position 9 is important for SP
activity [10,28,41], and for recognition discrimination between
the two binding sites associated to the NK-1 receptor [44].
Interestingly, septide, an equally potent Nk1r selective agonist,
has a proline residue in the position corresponding to Gly 9 of SP
[1], which could also induce a distortion in this region of the
peptide. It is generally postulated that this hinge at position 9
favors an extended conformation (similar to a polyproline II
helix or a b strand) of residues 9–11 that positions the C-terminal
in a fixed and specific conformation which is critical for receptor
recognition and agonist activity [38,41,44]. Studies with SP
containing a proline at this position only report a slight decrease
in IC50, from 2.7 to 1.8 nM compared to the native peptide [43].
Our finding that substitution at Gly-9 with TOAC results in a
100-fold decrease in both affinity and efficacy, strongly
suggests TOAC at Gly-9 interferes with the ability of the peptide
to adopt its native conformation in the Nk1r binding pocket. Our
simulations show that Gly-9 possesses some inherent structural
flexibility that allows it to adopt different conformations,
between roughly a a-helical and a left-handed helix. Interestingly, substitution of Gly-9 by the spin label TOAC introduces a
significant structural change in SP. While TOAC-9 is still able to
adopt the left-handed helical conformation, its conformational
space is extended to regions resembling PP-II. It is very likely that
these alternative conformations do not allow the rest of the Cterminal region of SP to adopt the conformation required for
proper ligand–receptor interaction, thus hindering the pharmacological properties of 9-TOAC SP. Therefore our results
support the idea that position 9 sets the end of the central helical
part of SP, introducing a specific distortion that is important for
SP activation of Nk1r. This structural feature can be introduced
by either a Gly or Pro, but not by the spin label TOAC. Substitution
at position 9 results in a destabilization of the active conformation of the C-terminal region of substance P, which would
explain the observed experimental results.
4.3.
4-TOAC does not disrupt the structure and
pharmacological profile of SP
Our pharmacological data (Figs. 1 and 2) show that TOAC
substitution at position 4 does not affect the high-affinity SP
binding to the NK1r or its ability to activate the receptor. Our
simulations reveal the ultimate structural reason for this
behavior. As seen in Fig. 5, labeling at position 4 does not affect
the structural properties of SP. In our SP model, Pro-4 is ahelical, in agreement with spectroscopic [38], and NMR studies
[41,44]. The analysis of the amino acid distribution in a-helices
of globular proteins [27], shows that Pro is the most favored
residue in the position immediately following the N-terminal
residue of an a-helix. At this location, Pro facilitates the
formation of hydrogen bonds involving the side chains of
residues at the N-term, thus acting as a helix starter. The
simulations confirm that the TOAC label acts as a helix
stabilizer, as the conformational space of this residue is tightly
clustered around the a-helical region (Fig. 5, bottom-left
panel). Therefore, in this case the strong structural constraints
imposed in the peptide backbone by the spin label do not alter
the structure of SP, which is translated in the pharmacological
profile of 4-TOAC SP being very similar to the wild type
peptide.
Interestingly, previous studies have shown that NKA and
septide tachykinins, which both lack the Pro-4 of SP, are
capable of high-affinity binding on the Nk1r [22,31]. Cascieri
et al. [9], suggested that higher selectivity of SP for the Nk1
receptor over Nk2 and Nk3 can be modulated by substitutions
at position 4, though this is accounted by increases in the
affinities measured for other receptor subtypes rather than a
loss in affinity for Nk1r. It has been suggested that other
tachykinins (NKA, NKB, septide) bind to Nk1r via alternative
ligand arrangements [6,31,57], Thus we cannot rule out that 4TOAC SP is coordinated in a manner distinct from native SP,
even though its binding affinity is relatively unchanged.
However, as discussed our modeling data would suggest that
there has been minimal deviation in the SP structure from the
introduction of TOAC at this position.
4.4.
4-TOAC SP is an excellent reporter for EPR
experiments on Nk1r
Radioligand binding data show that GPCRs undergo a shift
from a high to low affinity state when the G-protein
dissociates from the ternary complex that is thought to be
formed between the ligand, receptor and G-protein
[8,13,36,53]. In this work, we investigated the G-proteininduced conformational changes that the NK1 receptor
undergoes upon binding of TOAC labeled SP using membranes
isolated from NK1GFP expressing cells. Structural changes in
the receptor during activation will be coupled to structural
changes in the ligand, which can be measured by EPR. Since
the TOAC probe is integrated into the backbone of the peptide,
it serves as a more sensitive reporter of the receptor structural
changes than do larger probes attached to an amino acid side
chain. Consistent with its higher binding affinity, we were able
to detect a stronger signal for the 4-TOAC SP than for the 9TOAC SP. Thus, a significant fraction of the 9-TOAC SP is lost
during the membrane wash step following peptide incubation.
To test whether disruption of the ternary complex of
receptor, ligand and G protein is reflected by a change in the
binding and dynamics of the peptides observed by EPR, we
compared the incubation of membranes and TOAC SP with the
same mixture containing GTPgS. Following the wash step, the
peptides 29 (2008) 1919–1929
amount of signal obtained for the 9-TOAC SP in the presence of
GTPgS was negligible, thus an evaluation of peptide dynamics
based on EPR line shape changes was not possible. When we
added GTPgS to the membrane incubation containing 4-TOAC
SP, the TOAC spectrum becomes markedly sharper, suggesting the entire population of ligand has become less tightly
bound. Since we are unsure whether the level of endogenous G
protein is sufficient to modulate the level of over expressed
Nk1r, we would anticipate that the magnitude of the change
may be more pronounced in a stable cell line expressing the
receptor and G-protein at stoichiometric amounts. As shown
previously by Scatchard analysis, the stable cell line expressing the receptor does exhibit high and low affinity binding,
however, over expression of the receptor leads to much of the
receptor being unoccupied with G-protein [49]. Thus in the
absence of GTPgS, the spectrum of 4-TOAC SP is likely a
combination of peptides bound at high and low affinity states,
whereas the GTPgS spectrum is predicted to be dominated by
peptides bound at the low affinity state.
In summary, we report the first EPR detection of TOACsubstituted peptides binding to a GPCR in native membranes.
While both the 4- and 9-TOAC SP analogues retain Nk1r binding
and activation, limitations in instrumental sensitivity preclude
our ability to calculate distinct correlation times for the spectral
components apparent in the low- and high-affinity states of the
Nk1r protein. Future advances in resonator design and the
emerging application of higher microwave frequencies will
yield significant benefits in this regard [5,23]. Also, the
simulation of EPR spectra using computational approaches
[7], will allow a deeper understanding of the structural changes
encoded in the spectra. In addition to using TOAC-containing
agonists for probing Nk1r conformational changes associated
with G-protein interaction, these new derivatives provide
further insight into the structural requirements for both SP
affinity and specificity. Given the availability of a functional
Cys-engineered Nk1r [16], it is now possible to target nitroxide
spin labels near the putative SP binding site [38]. Thus, spin
coupling between a label located on the receptor and TOAClabeled SP ligands will provide a direct experimental approach
for the nature of SP binding as well as the accompanying
conformational shifts upon receptor activation.
Acknowledgments
We thank Dr. Mark A. Simmons, in whose laboratory the
calcium mobilization studies were conducted with the support
of NIH grant NS25999. This investigation was conducted in a
facility constructed with support from Research Facilities
Improvement Program Grant Number C06 RR-12088-01 from
the National Center for Research Resources, National Institutes of Health. XD research is supported by the Ministerio de
Educación y Ciencia (Spain), through the Juan de la Cierva
programme.
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