Abstract
The adenosine A2A receptor (A2AR) engages several G proteins, notably Go and its cognate Gs protein. This coupling promiscuity is facilitated by a dynamic ensemble, revealed by 19F nuclear magnetic resonance imaging of A2AR and G protein. Two transmembrane helix 6 (TM6) activation states, formerly associated with partial and full agonism, accommodate the differing volumes of Gs and Go. While nucleotide depletion biases TM7 toward a fully active state in A2AR–Gs, A2AR–Go is characterized by a dynamic inactive/intermediate fraction. Molecular dynamics simulations reveal that the NPxxY motif, a highly conserved switch, establishes a unique configuration in the A2AR–Go complex, failing to stabilize the helix-8 interface with Gs, and adoption of the active state. The resulting TM7 dynamics hamper G protein coupling, suggesting kinetic gating may be responsible for reduced efficacy in the noncognate G protein complex. Thus, dual TM6 activation states enable greater diversity of coupling partners while TM7 dynamics dictate coupling efficacy.
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Data availability
All data from this study are included in Source data files provided with this paper. Requests for further information, resources and reagents should be directed to and will be fulfilled by the lead contacts, Scott Prosser (scott.prosser@utoronto.ca), Louis-Philippe Picard (louisphilippe.picard@utoronto.ca) or Adnan Sljoka (adnan.sljoka@riken.jp).
Change history
28 August 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41589-024-01732-z
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Acknowledgements
This work was supported by the Canadian Institutes of Health Research (CIHR) Operating Grant no. MOP-43998 to R.S.P.; the MEXT/JSPS KAKENHI grant nos. JP19K23721 and JP23K14154 D.P.T. and grant nos. JP19H03191, JP22H04745, JP23H02445 and JP23H02445 to A.K.; and the MEXT ‘Program for Promoting Research on the Supercomputer Fugaku’ (Simulation- and AI-driven next-generation medicine and drug discovery based on ‘Fugaku’, grant no. JPMXP1020230120) to D.P.T. A.S. was supported by the RIKEN International Collaboration Program grant. S.K.H. was supported by an Alexander Graham Bell Canada Graduate Scholarship-Doctoral from NSERC. A.S. was supported by CREST, Japan Science and Technology Agency (JST), Japan, grant no. JPMJCR1402. We thank M. Bouvier for providing constructs for BRET studies, D. Pichugin for technical assistance and maintenance of the NMR spectrometer at the Center for Magnetic Resonance Research, University of Toronto Mississauga, and the UTM’s Office of the Vice-Principal, Research (OVPR), for providing research infrastructure needed for this research. We also thank S. Furness (U. Queensland) for helpful suggestions. This work also used computational resources provided by the Institute for Solid State Physics, the University of Tokyo, the Research Center for Computational Science, Okazaki, Japan (Project: 23-IMS-C045), and the supercomputer Fugaku through the HPCI System Research Project (Project IDs: hp220107, hp230077 and hp240024) and the RIKEN Center for Computational Science (Project ID: hp230216).
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L.-P.P., A.O. and R.S.P. designed the research. L.-P.P., A.O., Z.Q. and S.K.H. performed protein expression and purification. L.-P.P. performed BRET and hydrolysis experiments. L.-P.P. and A.O. performed NMR experiments. D.P.T. and S.H. performed MD simulations under the supervision of A.K. A.K. designed and supervised analysis of molecular modelling, MD simulations, PCA analysis and related analysis. A.T. performed MC simulations under the supervision of A.S. K.T. assisted with simulations. A.S. performed RTA analysis. A.S. designed and supervised computational analysis, M.C. simulations and rigidity theory analysis. L.-P.P., A.O., D.P.T., A.K., A.S. and R.S.P. prepared the manuscript. R.S.P. supervised the project.
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Extended data
Extended Data Fig. 1 A2AR coupling to multiple G proteins and corresponding Bioluminescence Resonance Energy Transfer (BRET) and 19F NMR response to Gs and Go associated with TM6.
(a) Schematic representation of the BRET sensor, showing activation through the addition of an agonist (yellow triangle) and the corresponding dissociation of the α and βγ subunits, allowing for formation of the GRK-GFP and βγ-RLuc BRET pair. (b) BRET-based assay showing coupling of A2AR to Gs and Go upon agonist (NECA) stimulation. Data represent mean +/− SEM of n = 4 independent experiments. (c) Overlay of inactive and mini-Gs bound active A2AR crystal structures, illustrating the reorientation of TM6. (d) 19F NMR of TM6 labeled A2AR. The A2AR inactive ensemble (S1TM6 and S2TM6) is distinguished by a salt bridge between R1023.50 and E2286.30 that is either intact (S1TM6, −61.2 ppm) or broken (S2TM6, −60.9 ppm), where disruption of the salt bridge is necessary for reorientation of TM6 and activation. The A2AR activation ensemble is characterized by a preactivated state (A3), observed upon complexation of the apo receptor with GDP-bound G protein, and two distinct active states (A1 and A2) which become prominent upon removal of nucleotide. The nucleotide-free A2AR-Go complex is characterized by a lack of the A1 state, formerly associated with full agonism in the A2AR-Gs complex. All 19F NMR spectra are deconvoluted into a superposition of Lorentzian lines as shown. The light red line illustrates the residual error between the fitted and experimental spectra.
Extended Data Fig. 2 TM7-labeled A2AR reveals multiple intermediates and activation states.
a) 19F NMR reveals a wealth of functional states in a dynamic equilibrium. Complexation with Gs results in an activation ensemble, defined by A1TM7, A2TM7 and A3TM7. Removal of nucleotide subsequently shifts this activation ensemble toward A1TM7. In contrast, complexation with Go results in a partial shift to the activation ensemble, while multiple activation intermediates persist upon nucleotide removal. b) Overlay of inactive and active crystal structures of A2AR depict reorientation of TM7 upon complexation with mini-Gs. c–d) T2-filtered spectroscopy of the nucleotide-free complexes reveals sub-millisecond timescale dynamics of the intermediates, while the surviving (long-T2) activation states are identical for the nucleotide-free A2AR-Gs and A2AR-Go complexes. In this experiment, signal associated with states undergoing conformational exchange comparable to or faster than the refocusing frequency will become decoherent and not be fully refocused such that an exchange rate can be derived75. The overall effect is the removal from the apparent spectra of any such homogeneous line broadening. Here, a delay of 150 μs between refocusing pulses was used in the T2-filtered spectroscopy, corresponding to a refocusing frequency (1/4τ) of 1.67 kHz.
Extended Data Fig. 3 RMSD of heavy atoms from the initial structures during MD simulations.
(a) RMSD of heavy atoms associated with A2AR only for the A2AR-Gs complex. Note that the 10 different colors designate separate 1 µs trajectories for a total sampling time of 10 µs. (b) RMSD of A2AR heavy atoms for the A2AR-Go complex. (c) RMSD of heavy atoms of Gs in the A2AR-Gs complex (d) RMSD of Go in the A2AR-Go complex. RMSD is calculated after first superimposing structures to the initial energy minimized reference structure. In general, the simulated structures were judged to converge after 500 ns. Note that one trajectory undergoes a significant departure at ~ 500 ns (orange in d), indicative of a global rearrangement of Go. (e) Overlay of A2A-Gs in cyan (in (c) at 990 ns indicated by the arrow) and the outlier A2AR-Gs (shown in red in (c) at 990 ns indicated by the arrow). The difference is largely a consequence of rearrangement of the αN helix of Gα and the Ras domain. (f) Overlay of A2A-Go in magenta (in (d) at 990 ns indicated by the arrow) and the outlier A2AR-Go (shown in orange in (d) at 990 ns indicated by the arrow). Here, the outlier is distinguished by a reorientation of the αN helix as well as that of Gγ.
Extended Data Fig. 4 Evaluation of direct interactions in the A2AR-Gs and A2AR-Go complexes.
a) Connectivity map showing residues making direct contact with Gαs. Magenta and grey lines designate hydrogen bonds or hydrophobic contacts respectively. b) A2AR-Gαo connectivity map.
Extended Data Fig. 5 Calculation of binding free energies for the A2AR-Gs and A2AR-Go complexes by dPaCS-MD/MSM.
(a, c) The dissociation process of G proteins from A2AR shown by the inter-center of mass distances between A2AR and Gs/Go, d, as a function of dPaCS-MD cycle. The results for a) A2AR-Gs and c) A 2AR-Go. (b, d) The potential of mean force (PMF) as a function of d for (b) A2AR-Gs and (d) A2AR-Go obtained from the Markov State Model (MSM). The results of five trials (dots) and the average (thick lines) are shown. The PMF of the bound state is −22.1 ± 1.6 (A2AR-Gs) and −19.7 ± 1.5 (A2AR-Go) kcal/mol compared to that of the completely unbound states. Considering the volume correction of 1.8 ± 0.1 (A2AR-Gs) and 1.7 ± 0.2 (A2AR-Go) kcal/mol, the standard binding free energies are −20.3 ± 1.6 (A2AR-Gs) and −18.0 ± 1.5 (A2AR-Go) kcal/mol. (e, f) The implied time scale versus lag time plots of MSM for (e) A2AR-Gs and (f) A2AR-Go. The lag time of 50 ps was used to construct MSM.
Extended Data Fig. 6 A2AR-Gs and A2AR-Go have distinct features of the NPxxY motif and conformational dynamics.
a) MD simulations reveal an ensemble of NPxxY A2AR-Gs and A2AR-Go dihedral angle configurations in a nucleotide-free or GDP-bound states. b) Difference in Root Mean Square Fluctuations as a function of residue across A2AR between A2AR-Gs and A2AR-Go complexes during MC simulation. Note that cutoff energies of 1, 2, and 3 kcal/mol refer to the extent of sampling of conformers and give rise to predictably greater excursions in the Gs and Go complexes, particularly in the vicinity of ECL2 and ICL3. c) MC-derived distances between R2938.48 and E378H5.24/G352H5.24.
Extended Data Fig. 7 Principal component analysis (PCA) of A2AR conformations sampled by 10 μs MD simulations.
Projections onto the space spanned by the first two principal components (PCs) and conformational changes along these PCs, showing the distributions of the snapshots from each cluster with different color. The stars indicate the representative structures of the most populated clusters (Cluster 1) and the crosses signify the representative structures from the rest of the clusters. The x- and y-axes are scaled by the standard deviations (σ) of PC1 and PC2 (1.0 and 0.7 Å for A2AR-Gs and 1.1 and 0.8 Å for A2AR-Go) (a) Superposition of A2AR-Gs structures (right) and corresponding PCA mapping (left). Structures were generated by moving the average structure at (PC1, PC2) = (0,0) from −0.5 σ to 0.5 σ along PC1 or PC2. (b) Superposition of A2AR-Go structures (right) and corresponding PCA mapping (left).
Extended Data Fig. 8 The volume of the H5-helix dictates TM6 position.
a) Superposition of 56 Gs coupled receptors(orange) and 15 Go coupled receptors (blue) shows TM6 adopts a more outward orientation by 2 Å in the case of Gs. b-c) H5 volume of Gs (b) and Go (c) on all structures shown in a protein modeled by MD simulations with volume calculations for the last 5 and 8 amino acids and full H5 on all structures showing a bigger volume for Gs.
Extended Data Fig. 9 Validation of Gα constructs.
a-b) GloSensor assays of A2AGs and A2AGo heterotrimers, utilizing the 19F-labeled Δ5 Gαs and Δ5 Gαo constructs. Data are presented as mean values +/− SEM of n = 4 independent experiments. c) Native tryptophan residues and the conserved FH5.08 are overlayed between GDP-bound Gαs (PDB: 6eg8, dark red and brown) and GTPγS-bound Gαs (PDB: 1AZT, red and orange). d) 19F NMR spectra of 5FW-enriched Gαs. Individual unassigned resonances are resolved (P1S-P4S) for each of the four native tryptophan residues for the WT Gαs (dark red spectra). GTPγS substitution (light red spectra) produces no changes aside from a downfield shift in resonance P2S. The mutant F362WG.H5.08Gαs produces spectra with an additional severely broadened resonance (P5S, −128.25 ppm). GTPγS for GDP substitution (orange and brown spectra, respectively) reveals no large-scale changes in H5 helix topology related to the bound nucleotide, however, significant line broadening suggests local exchange processes at the H5 helix. e) Native tryptophan residues and the conserved FH5.08 are shown in the GDP-bound state (PDB: 3c7k, Blue and teal, respectively). f) 19F NMR spectra of 5FW-enriched Gαo. Unassigned resonances for the two native tryptophan residues are resolved for the WT Gαo in the GDP-bound (dark blue) and GTPγS-bound (light blue) states (P1O, P2O) where, only one tryptophan is sensitive to nucleotide exchange. Finer resolution of minor state conformers in the GDP-bound state alongside the dominant peak P2O indicate plasticity around the nucleotide core which is abrogated upon exchange for GTPγS. Mutant F336WG.H5.08 Gαo, similarly to Gαs, is insensitive to the presence of either GDP (teal spectra) or GTPγS (aqua spectra). The results reflect the higher basal activity of Go, where the dominant resonance of the nucleotide sensitive tryptophan associated with GTPγS binding is also dominant in the GDP-bound state.
Extended Data Fig. 10 Allosteric pathways in the A2AR-Gs and A2AR-Go complexes and commonalities with the serotonin-G protein complexes.
(a-b) Predicted allosteric pathway propagating through the G-protein upon rigidification of the nucleotide binding pocket in the A2AR-Gs (a) and A2AR-Go (b) complexes. (c-d) Predicted allosteric pathway propagating through the receptor in the 5HT4-Gs (c) and 5HT4-Gi (d) complexes, upon rigidification of the agonist binding contacts.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2.
Supplementary Video 1
Video of the A2AR-Gs complex and associated principal components: PC1, PC2 and PC3. Significant conformational changes are observed within ECL2, ICL3 and NPxxY motifs through the trajectories.
Supplementary Video 2
Video of the A2AR-Go complex and associated principal components: PC1, PC2 and PC3. Significant conformational changes are observed within ECL2, ICL3 and NPxxY motifs through the trajectories.
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Picard, LP., Orazietti, A., Tran, D.P. et al. Balancing G protein selectivity and efficacy in the adenosine A2A receptor. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01682-6
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DOI: https://doi.org/10.1038/s41589-024-01682-6
