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Supporting Information for
A GTPase chimera illustrates an uncoupled nucleotide affinity
and release rate, providing insight into the activation
mechanism
Amy P Guilfoyle,
Tourle,
1,2
1,2
Chandrika N Deshpande,
4
1,2
Josep Font,
5,*
1,2
Gerhard Schenk, Megan J Maher, and Mika Jormakka
1
3
Miriam-Rose Ash, Samuel
1,2,*
2
Structural Biology Program, Centenary Institute, Locked Bag 6, Sydney, New South Wales 2042, Australia; Faculty of
3
Medicine, Central Clinical School, University of Sydney, Sydney, New South Wales 2006, Australia; Department of
4
Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark; School of
5
Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland 4072, Australia; La Trobe
Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
*Correspondence to m.maher@latrobe.edu.au or m.jormakka@centenary.org.au
METHODS
Protein preparation:
The gene encoding the ChiNFeoB protein was generated by substitution of residues 150
to 158 inclusive from the G5 loop and succeeding α-helix in E. coli NFeoB for the equivalent
residues (326 to 335) from the eukaryotic GNAi1 (Giα1; Gene ID 2770) using overlapping
extension PCR (Fig. 1e). The construct was cloned into the pGEX-4T-1 vector for expression as a
GST fusion protein with thrombin recognition site.
Overexpression was carried out in BL21 (DE3) cells under ampicillin selection (50 µg
ml-1) in Luria Broth (LB) and purified as previously described for E. coli NFeoB protein (2).
Briefly, cells were incubated at 37 °C until OD600 reached 0.6, the temperature was reduced (25
°C, 20 min) and expression initiated with the addition of 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG, Astral). After 4 hours of expression the cells were harvested by
centrifugation (6,000 × g, 15 min, 4 °C), and resuspended in Buffer 1 (20 mM Tris pH 8.0, 100
mM NaCl). Cells were lysed in a high- pressure homogeniser (13 kPsi, Emulsiflex-C3, Avestin
Inc.), and cellular debris was removed by centrifugation (86,000 × g, 45 min, 15 °C). The
supernatant containing soluble chimera protein was purified using GSH-affinity resin
(Glutathione sepharose 4B resin, GE healthcare) at 4 °C. To remove the GSH-tag, the protein was
incubated (48 hours, 37 °C) with thrombin (60 units) and CaCl2 (1.25 mM). The cleaved protein
was eluted from the resin in Buffer 1 (5-10 column volumes) and concentrated to approximately 1
mL. The concentrated protein was further purified by gel filtration chromatography (Superdex 75
HiLoad 26/600, GE Healthcare) in Buffer 1. Purified chimera protein was buffer exchanged into
20 mM Tris pH 8.0, concentrated to approximately 10 mg ml-1, and was stored at -20 °C until
further use.
GTPase activity measurements:
The GTP hydrolysis rate for the ChiNFeoB protein was compared with WTNFeoB using
a Malachite Green Phosphate Assay (BioAssay Systems). Protein (0.3 µM) was incubated with
GTP (400 µM) in buffer (20 mM Tris pH 8.0, 200 mM KCl) at 37 °C. Hydrolysis was initiated
with the addition of MgCl2 (5 mM) and proceeded for an average 3.5 hours. Aliquots were
removed at frequent intervals and mixed with the Malachite Green reagent in a 4:1 ratio as per
manufacturer specifications. Color was developed for 30 min at room temperature prior to
absorbance measurements (620 nm) on a POLARstar Omega microplate reader (BMG Labtech)
in a 96- well plate (Greiner Bio-One). The enzyme turnover number (kcat) was determined for
wtNFeoB and ChiNFeoB at 37 ºC by means of linear regression and all hydrolysis assays were
performed in triplicate.
Stopped-Flow Fluorescence Assays:
The binding and release rates of fluorescent nucleotides by wtNFeoB and ChiNFeoB
were analyzed using stopped flow fluorescence assays. To determine release rates (koff), protein
(10 µM) was incubated with the fluorescent nucleotide mant-GDP (0.5 µM), in stopped flow
buffer (20 mM Tris pH 8.0, 100 mM NaCl, 100 mM MgCl2) for 30 min at room temperature.
Equal volumes of the protein-mant-GDP mix and GTP (1 mM) in stopped flow buffer were
rapidly mixed into a 100 µl optical cell of a pneumatically driven stopped flow apparatus (SMV17MV, Applied PhotoPhysics). The mant group was excited at 360 nm and fluorescence was
monitored through a 405 nm cut-off filter. Similarly, nucleotide-binding rates (kobs) were
determined by rapidly mixing protein (2.5- 80 µM) with the fluorescent non-hydrolyzable GTP
analogue, mant-GMPPNP (1 µM), in the stopped flow apparatus. All data reported are averaged
from 7-10 independent experimental traces performed under identical conditions. Reactions were
performed at 20 °C.
Isothermal Titration Calorimetry:
Isothermal Titration Calorimetry (ITC) was used to measure the GDP binding affinity of
wtNFeoB and ChiNFeoB proteins. Protein (approximately 0.1 mM) in buffer (20 mM Tris pH
8.0, 100 mM NaCl) was equilibrated for 1 min at 25 °C with stirring (1000 rpm) in the sample
cell of a MicroCal iTC200 Isothermal Titration Calorimeter. GDP (2.5-5 mM) was titrated into the
sample cell in 0.5-2 µl injections over 0.8 sec with 150 sec spacing between injections. Power
input (µcal sec-1) required to maintain equal temperatures between the sample and reference cells
in response to each addition of ligand was plotted versus time (min). The data was integrated and
plotted versus the molar ratio of ligand to protein. Non-linear regression was used to obtain the
thermodymanic parameters (including GDP binding affinity, Ka). Data were fitted to a one site
binding model using the Origin 7 Software (MicroCal) to obtain stoichiometry (N), enthalpy
(ΔH), entropy (ΔS), and the association constant (Ka). The dissociation constant (Kd) was
calculated from the equation Kd= 1/Ka. All reported values are the average of three or more
independent titrations. Due to interdiffusion of the solutions during the insertion of the syringe
into the sample chamber, the first injection is not useful for analysis and was omitted from all
calculations.
Protein Crystallization and Structure determination:
Crystals of the GDP-bound ChiNFeoB protein were grow via hanging drop vapor
diffusion from protein (20 mgmL-1) incubated with GDP (2:1 molar ratio of GDP: protein)
overnight at 4 °C. Crystals reached maximum size after three days grown from 1:1 ratio of
protein (1 µL) to reservoir solution (1 µL, 22 % PEG 3350, 0.1 M Bis Tris Propane pH 6.5 and
0.2 M Sodium formate) incubated at 20 °C. The chimera-GDP crystals were cryoprotected in
reservoir solution containing 30 % glycerol and flash cooled in liquid nitrogen. Data were
collected using a Rigaku RU-200 rotating anode generator and recorded on a MAR345 image
plate detector. Diffraction data were processed and scaled using HKL2000 (3,4). Cell dimensions
and data collection statistics are presented in Table S1. Chain A from the structure of wild type
E.coli NFeoB (3HYR residues 1-260) with the G5 motif (150 to 158 inclusive) and water atoms
removed and with Se-Met residues replaced by Met was used to solve the molecular replacement
solution using Phaser to a resolution of 2.2 Å (5). The resulting model was refined by iterative
cycles of amplitude based twin refinement (using twin operators H, K, L and –H, K, -L with
estimated twin fractions of 0.502 and 0.498 respectively) within REFMAC (6,7), interspersed
with manual inspection and correction against calculated electron density maps using COOT (8).
Table S1 – Data processing and refinement statistics
Data collection
Wavelength (Å)
Space group
Unit cell parameters (Å)
Resolution (Å)
Total reflections
1.5412
P41
a=b=48.5; c=233.4
50.0 - 2.20 (2.28-2.20)
Unique reflections
Completeness (%)
26357
Multiplicity
(I/σ(I))
6.8
Rmerge#
0.044 (0.224)
Unique reflections
Completeness (%)
Rwork§
Rfree
<Protein B factor> (Å2)
No. of protein molecules per
asymmetric unit
R.m.s.d bonds (Å)
R.m.s.d angles (°)
Ramachandran plot statistics+
Favoured (%)
Allowed (%)
PDB code
26345
99.4 (95.4)
0.232
0.276
38.76
2
1131277
97.1 (73.6)
36.8 (5.6)
0.004
0.900
96.84
3.16
4R98
*Values in parentheses are for the highest resolution shell.
#
Rmerge = S|Ih - <Ih>|/S<Ih>
§
Rwork = Shkl | |Fobs| - |Fcalc| |/Shkl |Fobs|
+
As calculated by MolProbity
Supporting References
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21752-21758.
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laboratory manual, 3rd. ed., Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory,
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oscillation mode. in Methods in Enzymology (Charles W. Carter, Jr. ed.), Academic Press. pp
307-326.
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