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 [email protected] or [email protected] 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 1. Posner, B. A., M. B. Mixon, M. A. Wall, S. R. Sprang, and A. G. Gilman. 1998. The A326S Mutant of Gialpha1 as an Approximation of the Receptor-bound State. J. Biol. Chem. 273: 21752-21758. 2. 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