Protein cloning, expression, and purification
Cloning of the wild type Gsα construct has been described previously (Huang et al., 2022). The human GNAS2 gene was subcloned into a pET15b vector immediately downstream of an open reading frame for hexahistidine-tagged maltose binding protein (MBP) and tobacco etch virus (TEV) cleavage site. The ΔCys construct (C3S, C200T, C237S, C359I, C365A, C379V) and additional single-cysteine mutants (ΔCys-K34C, ΔCys-R61C, ΔCys-Y358C, ΔCys-E370C) were generated via site-directed mutagenesis using the pET15b-Gsα wild type or ΔCys plasmid as a template. All mutations were confirmed by DNA sequencing at the Centre for Applied Genomics (TCAG), the Hospital for Sick Children (Toronto, Canada).
The expression and purification of Gsα (identical for the wild type and all mutant constructs) has been described previously (Huang et al., 2021). Briefly, Escherichia coli (E. coli) BL21 (DE3) cells containing the vector carrying the desired Gsα construct were grown in LB Miller medium at 25 °C to an optical density at 600 nm (OD600) of 0.3. Cells were induced overnight at 19 °C with isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 50 µM, then harvested by centrifugation at 6,000 g. Cell pellets were resuspended lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole, 50 µM GDP, 2 mM MgCl2, 0.5 mM TCEP, 5 mM 6-aminocaproic acid, 5 mM benzamidine, 0.4 mg/mL lysozyme, 2 µg/mL DNase I, 10% glycerol) and lysed via sonication. The lysate was centrifuged at 20,000 g for 30 minutes, and the resulting supernatant was incubated with TALON resins (Takara Bio) for 3 h at 4 °C with gentle mixing. For 19F labeling, the TALON resins were washed with 3 bed-volumes of labeling buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP, 10% glycerol), then resuspended in 2 bed-volumes of labeling buffer containing 200 µM 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA, Apollo Scientific). The reaction was allowed to proceed overnight at 4 °C with gentle mixing. A second aliquot of BTFMA was added and incubated for 6 h prior to loading the resin onto a gravity column. The resins were washed with lysis buffer containing 20 mM imidazole, then eluted with elution buffer (50 mM sodium phosphate (pH 8.0), 100 mM NaCl, 50 µM GDP, 2 mM MgCl2, 0.5 mM TCEP, 10% glycerol, 250 mM imidazole). The eluted MBP-Gsα was buffer exchanged using a centrifugal filter to remove imidazole, then incubated with 10 µg/mL of TEV protease (produced in-house) at 4 °C overnight without agitation. The sample was loaded onto a Ni-NTA column and the flow-through was collected and purified on a HiLoad 16/600 Superdex 200 prep grade size exclusion column equilibrated with 50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 5 µM GDP, and 10% glycerol. The eluted fractions containing Gsα were pooled, concentrated, and buffer-exchanged to final storage buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 100 µM GDP, 10% glycerol). The proteins are flash-frozen in liquid nitrogen and stored at -80 °C until needed.
Gβγ, and A2AR were expressed and purified as previously described (Huang et al., 2021). Empty nanodiscs were prepared as previously described (Huang et al., 2022). Both the A2AR and the empty nanodiscs were reconstituted using the MSPΔH5 membrane scaffold protein (Hagn et al., 2013) and a 3:2 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The protein or nanodisc samples were flash-frozen in liquid nitrogen and stored at -80 °C in the following storage buffers until use. For Gβγ: 50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.0125% n-Dodecyl β-D-maltoside (DDM), 100 µM TCEP, 100 µM GDP, 10% glycerol. For A2AR and empty nanodiscs: 50 mM HEPES, pH 7.4, 100 mM NaCl.
The purified protein stocks, stored in their respective storage buffers as described above, were combined such that the final NMR sample contained 30-80 µM BTFMA-labeled Gsα. The samples were supplemented with a final concentration of 10% D2O and 20 µM sodium trifluoroacetate (TFA) as the 19F chemical shift reference. 1.2-fold excess of Gβγ, 3-fold excess of A2AR, or 3.5-fold excess of empty nanodiscs, relative to the Gsα concentration, were added where applicable. Where needed, the sample also contained a final concentration of 100 µM GDP (GDP or GDP-AlF4- conditions), 50 µM AlCl3 and 5 mM NaF (GDP-[AlF4]- conditions), 2 U/mL apyrase (nucleotide-free conditions), 2 U/mL apyrase and 1 mM GTPγS (GTPγS conditions), or 1 mM NECA (A2AR + NECA conditions). We note that by itself, nucleotide-free Gα is unstable and tend to aggregate within an hour at concentrations suitable for an NMR experiment. In the presence of membrane, Gβγ, or receptor, samples are stable for at least several hours to several days depending on the mutant and the temperature of storage. We also note that due to large quantities of proteins that would be required, NMR of the Gsαβγ+A2AR+NECA+GTPgS condition was only carried out for the K34C construct. All samples were prepared in Shigemi tubes with a final volume of 200-350 µL. NMR experiments were acquired at 20 °C on either a 600 MHz Varian Inova spectrometer equipped with a 19F-tunable triple-resonance cryoprobe or a 500 MHz Varian Inova spectrometer equipped with a 5 mm room temperature inverse HFX probe. A typical 1D fluorine experiment included a 100-300 ms recycle delay, a 5.5-7 μs (45°) excitation pulse, and a 500-600 ms acquisition time. Each experiment was acquired using 24,000-200,000 scans, yielding a S/N of approximately 30-100. Spectra were processed using MestReNova (Mestrelab Research S.L.) employing chemical shift referencing (-75.6 ppm for TFA), zero filling, baseline correction, and exponential apodization equivalent to a 20-30 Hz line broadening.
GTP hydrolysis assays
GTP hydrolysis assays were performed using the GTPase-GloTM assay kit (Promega, Madison, WI, USA) following the manufacturer’s protocol (Mondal et al., 2015). Reactions were carried out at room temperature in a buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 1 μM GDP, and 4 μM GTP. Each reaction contained either Gsα alone (250 nM), Gsαβγ (250 nM Gsα + 300 nM Gβγ), or Gsαβγ with A2AR (250 nM Gsα + 300 nM Gβγ + 250 nM A2AR + 100 µM NECA). All reaction mixtures were pre-incubated for 1 hour prior to the addition of GTP, which initiated the reaction. A control was also carried out with only buffer and no G protein. After 90 minutes, unhydrolyzed GTP was converted to ATP and detected using a reagent containing luciferase. The resulting luminescence is proportional to the amount of unreacted GTP and measured using a TECAN Spark multi-mode plate reader with an integration time of 1 minute. The amount of GTP hydrolysis for each condition (x) can be represented by its difference in luminescence with the control:
Computational rigidity-transmission allostery predictions
Starting with the structure of GDP-bound Gsαβγ heterotrimer (PDB: 6EG8), we measured the allosteric propensity from the GDP binding pocket across the complex by applying rigidity-transmission allostery (RTA) analysis (Sljoka, 2021). The RTA algorithm, whose details have been previously described (Sljoka, 2021; Ye et al., 2018), is a computational tool based on the mathematical rigidity theory which identifies allosteric networks within structures of proteins and protein complexes (Jacobs et al., 2001; Sljoka, 2021; Whiteley, 2005, Ye 2018, Mehrabi 2019). The algorithm was verified with NMR chemical shift data and predicts how changes in the conformational flexibility of one region in the protein are transmitted to distal sites by quantifying the resulting regiospecific change in conformational degrees of freedom. We applied the RTA algorithm on the GDP-bound Gsαβγ heterotrimer by rigidifying the bound GDP. The available conformational degrees of freedom were quantified for each residue before and after GDP rigidification. The resulting change in degrees of freedom was then extracted for each residue and represents the extent of allosteric transmission from the GDP binding pocket.