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Structure of the adenosine A2A receptor bound to an engineered G protein

Nature volume 536, pages 104–107 (2016)Cite this article

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An Erratum to this article was published on 14 September 2016

Abstract

G-protein-coupled receptors (GPCRs) are essential components of the signalling network throughout the body. To understand the molecular mechanism of G-protein-mediated signalling, solved structures of receptors in inactive conformations and in the active conformation coupled to a G protein are necessary1,2. Here we present the structure of the adenosine A2A receptor (A2AR) bound to an engineered G protein, mini-Gs, at 3.4 Å resolution. Mini-Gs binds to A2AR through an extensive interface (1,048 Å2) that is similar, but not identical, to the interface between Gs and the β2-adrenergic receptor3. The transition of the receptor from an agonist-bound active-intermediate state4,5 to an active G-protein-bound state is characterized by a 14 Å shift of the cytoplasmic end of transmembrane helix 6 (H6) away from the receptor core, slight changes in the positions of the cytoplasmic ends of H5 and H7 and rotamer changes of the amino acid side chains Arg3.50, Tyr5.58 and Tyr7.53. There are no substantial differences in the extracellular half of the receptor around the ligand binding pocket. The A2AR–mini-Gs structure highlights both the diversity and similarity in G-protein coupling to GPCRs6 and hints at the potential complexity of the molecular basis for G-protein specificity.

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Figure 1: Ligand binding and overall structure of the A2AR–mini-Gs complex.
Figure 2: Packing interactions between A2AR and mini-Gs.
Figure 3: Comparison of the A2AR–mini-Gs and β2AR–Gs complexes.
Figure 4: Conformational changes in A2AR upon G-protein binding.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic co-ordinates and structure factors for the A2AR–mini-Gs complex have been submitted to the Protein Data Bank (PDB) under accession code 5G53.

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Acknowledgements

This work was supported by a grant from Heptares Therapeutics Ltd., the ERC (grant EMPSI 339995) and core funding from the Medical Research Council (MC_U105197215 and MC_U105184325). We thank the beamline staff at the European Synchrotron Radiation Facility (beamlines ID23-2, ID30-A3 and ID29) and at Diamond Light Source (beamline I24). We also thank R. Henderson, A. Jazayeri and T. Flock for comments on the manuscript.

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Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

    Byron Carpenter, Rony Nehmé, Tony Warne, Andrew G. W. Leslie & Christopher G. Tate

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  1. Byron Carpenter
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  2. Rony Nehmé
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  3. Tony Warne
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Contributions

B.C. performed receptor expression, purification, crystallization, cryo-cooling of the crystals, data collection, data processing and structure refinement. T.W. helped with crystallization, data collection and data processing. R.N. performed the stability assays and pharmacological analyses on A2AR–mini-GS complexes. A.G.W.L. was involved in data processing and structure solution, refinement and analysis. Manuscript preparation was performed by B.C., A.G.W.L. and C.G.T. The overall project management was by C.G.T.

Corresponding authors

Correspondence to Byron Carpenter or Christopher G. Tate.

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Competing interests

This work was funded in part by Heptares Therapeutics Ltd. C.G.T. is a consultant and member of the Scientific Advisory Board for Heptares Therapeutics Ltd.

Additional information

Reviewer Information Nature thanks O. Ernst, K. Jacobson and K.-N. Klotz for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Pharmacological analyses of A2AR–mini-Gs complexes.

Competition assays were performed on A2AR expressed in HEK293 cell membranes with the agonist NECA competing for the binding of radiolabelled inverse agonist 3H-ZM241385. Experiments were performed in the presence of either 100 mM KCl (a, b), 100 mM NaCl (c, d) or 500 mM NaCl (e, f) to confirm the similar behaviour of mini-Gs with heterotrimeric Gs with nanobody Nb35 for stabilization of the complex. Results are summarized in the Table (g). All error bars represent the s.e.m. for at least three independent experiments performed in duplicate. Comparisons of data in b, d and f were performed using an unpaired t-test with significance denoted by asterisks: ***P < 0.0001; **P < 0.01; *P < 0.1; not significant (NS) P > 0.1.

Extended Data Figure 2 Thermostability of detergent-solubilized 3H-NECA-bound A2AR in the presence or absence of mini-Gs.

Unpurified A2AR was solubilized in detergent at the following concentrations: a, DDM, 0.1%; b, DM, 0.13%; c, OG, 0.8%. Samples were heated for 30 min, quenched on ice and the amount of 3H-NECA bound determined. Data were analysed by nonlinear regression and apparent Tm values (transition temperature where 50% of the receptor is inactive) were determined from analysis of the sigmoidal dose–response curves fitted (d). Results represent the mean ± s.e.m. of two independent experiments, performed in duplicate.

Extended Data Figure 3 Omit maps for NECA and GDP.

a–f, Orthogonal views of omit map difference density for NECA in A2AR chain A (a, b), NECA in A2AR chain B (c, d) and GDP in mini-Gs chain C (e, f). The contour level is 2.5σ in panels a–d and 3.0σ in panels e and f. Figures were made using CCP4mg30.

Extended Data Figure 4 Electron density for the interface region of the A2AR–mini-Gs complex.

The backbones of A2AR and mini-Gs are shown in cartoon representation in light blue and magenta respectively. Side chains are shown in stick representation (carbon, light blue; oxygen, red; nitrogen, deep blue). The electron density of the final 2Fo − Fc map is shown contoured at 1.2σ. For clarity, transmembrane helices H5 and H6 and the corresponding electron density have been omitted. a, View showing the interaction between the C-terminal helix of mini-Gs and the CL2 loop of A2AR. b, View showing the interactions between side chains of the C-terminal helix of mini-Gs and three Arg residues of A2AR.

Extended Data Figure 5 Alignment of mini-Gs with GNAS2.

Comparison of amino acid residues in mini-Gs (chains C and D) within 3.9 Å of A2AR (green) in the A2AR–mini-Gs structure and the amino acid residues in bovine GNAS2 (P04896) within 3.9 Å of β2AR in the β2AR–Gs structure (turquoise). The CGN system is used for reference.

Extended Data Figure 6 Alignment of β2AR and A2AR amino acid sequences.

adrb2_human, human β2-adrenergic receptor; AA2AR_human, human adenosine A2A receptor; AA2AR chain A, chain A of the crystallized A2AR–mini-Gs structure; AA2AR chain B, chain B of the crystallized A2AR–mini-Gs structure. Residues in the receptors that are within 3.9 Å of either Gα in the β2AR–Gs complex or mini-Gs in the A2AR–mini-Gs complex are highlighted in turquoise or green, respectively. Key Ballesteros–Weinstein numbers are shown in blue and mutations in the crystallized A2AR to facilitate purification and crystallization are shown in red. Grey bars indicate the positions of α-helices in the β2AR–Gs structure, whereas red bars represent these regions in the A2AR–mini-Gs structure; where there is a discrepancy in helix length between chain A and B of A2AR, the bar is coloured pink.

Extended Data Figure 7 A conserved hydrophobic binding pocket at the receptor–Gαs interface.

The A2AR–mini-Gs complex was aligned to the β2AR–Gs complex via the receptors; A2AR, green; β2AR, turquoise; mini-Gs (purple); Gαs (grey).

Extended Data Figure 8 Comparison between receptor-bound mini-Gs and Gαs.

a–c, Three different views of an alignment of mini-Gs (chain C, purple) bound to A2AR with the GTPase domain of Gαs (grey) bound to β2AR. GDP bound to mini-Gs is depicted as a space filling model (carbon, yellow; oxygen, red; nitrogen, blue; phosphorus, orange). The α5 helix that interacts with the receptors is labelled.

Extended Data Figure 9 Comparison of the NECA binding site in the active-intermediate state compared to the mini-Gs-bound state.

The structure of NECA-bound A2AR (grey cartoon, with the carbon atoms of NECA also in grey) in the active-intermediate state was aligned with the structure of the NECA-bound A2AR–mini-Gs complex (rainbow colouration, with the carbon atoms of NECA in green). Key amino acid residues for both receptors are depicted (sticks; carbon atoms in the same colour as the respective receptor) that form hydrogen bonds (red dashed line) with either NECA or the associated water network (red spheres). Note that the water molecules depicted are from only the NECA-bound A2AR structure in the active-intermediate state, because the resolution of the A2AR–mini-Gs structure was insufficient to identify water molecules. Carbonyl oxygens are denoted by ‘co’ after the residue name.

Extended Data Table 1 Data collection and refinement statistics
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Carpenter, B., Nehmé, R., Warne, T. et al. Structure of the adenosine A2A receptor bound to an engineered G protein. Nature 536, 104–107 (2016). https://doi.org/10.1038/nature18966

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