Abstract
The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound β2 adrenergic receptor–Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.
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Acknowledgements
This work was supported by the Monash University Ramaciotti Centre for Cryo-Electron Microscopy, National Health and Medical Research Council of Australia (NHMRC) project grant APP1145420 and NHMRC program grant APP1055134. A.C., P.M.S. and D.W. are NHMRC Senior Principal Research, Principal Research and Career Development Fellows, respectively. A.G. and D.M.T. are Australian Research Council Discovery Early Career Research Fellows. L.T.M. is an Australian Heart Foundation Future Leaders Fellow.
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C.J.D.-J. developed the expression and purification strategy, performed virus production, insect cell expression, purification, and membrane-based pharmacological assays, negative-stain electron microscopy data acquisition/analysis and prepared samples for cryo-EM; M.K. performed sample plunging for cryo-EM, phase-plate imaging and data collection, electron microscopy data processing and analysis; D.M.T. developed the expression and purification strategy, assisted with biochemistry, structure refinement and validation and model interpretation; Y.-L.L. and S.G.B.F. developed the strategy to generate the dominant-negative Gαi2; R.D. and W.B. organized and developed the Volta phase-plate cryo-EM data acquisition strategy; H.V. organized microscopy time and provided oversight of image acquisition within the Monash EM facility; J.M.P. provided advice on microscope setup for phase-plate imaging and EM facility access within the Max Planck Institute; A.T.N.N. and J.A.B. performed whole-cell pharmacological assays; L.T.M. supervised whole-cell pharmacological assays; C.J.D.-J., A.T.N.N., J.A.B. and L.T.M. performed data analysis; M.K., D.M.T., Y.-L.L., S.G.B.F., L.T.M., D.W. and P.M.S. assisted with data interpretation and preparation of the manuscript; A.G. developed the expression and purification strategy, performed negative-stain electron microscopy, cryo-EM sample preparation, model building, refinement and validation; C.J.D.-J., A.G. and A.C. wrote the manuscript; P.M.S., A.G. and A.C. supervised the project.
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Extended data figures and tables
Extended Data Fig. 1 Expression and purification of the ADO–A1R–Gi2 complex.
a, Schematic of the haemagglutinin (HA) and Flag-tagged M4R-3C-A1R-3C-8×His construct. The most conserved residue for class A GPCRs (X.50 class A numbering34) are highlighted for each transmembrane domain in red. b, Purification step flowchart for the A1R–Gi2 complex. c, SDS–PAGE/western blot of samples obtained at various stages of A1R–Gi2 purification. A1R and the Gi2 heterotrimer were expressed separately in insect cell membranes. Addition of ADO initiated complex formation, which was solubilized by detergent. Solubilized A1R and A1R –Gi2 complex were immobilized on Flag antibody resin. Flag-eluted fractions were purified by size-exclusion chromatography (SEC). An anti-His antibody was used to detect Flag–A1R-His and Gβ-His (red) and an anti-Gi2 antibody was used to detect Gαi2 (green). d, SDS–PAGE/Coomassie blue stain of the purified complex concentrated from the Superdex 200 Increase 10/30 column. e, Left, representative elution profile of Flag-purified complex on Superdex 200 Increase 10/30 SEC. Right, SEC fractions containing A1R–Gi2 complex (within dashed lines) were pooled, concentrated and analysed by re-running on Superdex 200 Increase 10/30 column. All images and SEC profiles are representative of more than three independent experiments.
Extended Data Fig. 2 Pharmacology of the A1R construct and rationally chosen mutations.
a, b, Competition between the antagonist [3H]DPCPX and either unlabelled DPCPX (a) or ADO (b), in membranes expressing HA–Flag–3C-A1R-3C-8×His construct in the absence or presence of wild-type (WT) Gi2 heterotrimer or dominant-negative (DN) Gi2 heterotrimer. Data are normalized to [3H]DPCPX binding in the absence of unlabelled competitor, with nonspecific binding determined in the presence of 1 µM of the antagonist, SLV320. c, ADO-mediated binding of [35S]GTPγS as a measure of G-protein activation by the HA–Flag–3C-A1R-3C-8×His construct in High Five cells expressing receptor alone, or together with either wild-type or dominant-negative Gi2 heterotrimer. d, e, [3H]DPCPX competition assays (d) or inhibition of forskolin-stimulated cAMP accumulation (e), at the wild-type human A1R or two key alanine substitution mutations stably expressed in CHOFlpIn cells. f, Changes in agonist (NECA) affinity (Ki) from the experiments shown in d. g, Changes in NECA signalling efficacy corrected for receptor expression (τc), determined from the experiments shown in e. Parameter estimates are the mean ± s.e.m. determined from 3 (a–c) or 6–48 (d–g) independent experiments performed in duplicate. ****P < 0.0001 (compared with wild type; one-way analysis of variance (ANOVA), Dunnett’s post hoc test). Data for wild-type and N159A are replotted from Nguyen et al28.
Extended Data Fig. 3 Cryo-EM of the ADO–A1R–Gi2 complex.
a, Representative VPP cryo-EM micrograph (of 3,220 recordings) of the ADO–A1R–Gi2 complex. b, Reference-free 2D class averages of the complex in LMNG and CHS detergent micelles. c, Gold-standard Fourier shell correlation (FSC) curves, showing the overall nominal resolution at 3.6 Å. d, FSC curves for the final model versus the final map and the half maps for overfitting validation (see Methods).
Extended Data Fig. 4 Atomic resolution model of A1R transmembrane domains, the Gα protein α5-helix, ADO, and representative regions of Gβ and Gγ in the cryo-EM density map.
a, The molecular model is shown in stick representation and the cryo-EM map in mesh contoured at 0.06. b–d, A1R residues (b, c,) and Gαi2 α5-helix residues (d). The molecular model is shown in stick representation and the cryo-EM map in mesh contoured at 0.06.
Extended Data Fig. 5 Comparison of active and alternative inactive A1R (PDB code 5NS2) structure.
a–c, Side (a), extracellular (b) and cytoplasmic (c) view of the ADN–A1R–Gi2 structure (blue) compared to the inactive PSB36-bound A1R (grey). d, e, Active ADO–A1R (d) and inactive PSB36–A1R (e) receptor surfaces sliced to show binding site cavity. f, Orthosteric binding site of the active A1R–Gi2 complex with ADO (purple ball and sticks). ‘Toggle switch’ W2476.48 and residues within 4 Å of ADO are labelled and shown as sticks. Red rectangles highlight rotamer changes upon receptor activation. N, O and S atoms are coloured in blue, red and yellow, respectively. Dashed lines represent hydrogen bonds. g, GPCR motifs important for receptor activation (DRY motif, purple; NPXXY motif, blue; PIF motif, green).
Extended Data Fig. 6 Comparison of active A1R with active A2AR (PDB code 5G53) or agonist-bound ‘intermediate’ state A2AR (PDB code 2YDV).
a–j, Side views (a, f), extracellular views (b, g) and cytoplasmic views (c, h) of the active ADO–A1R–Gi2 structure (blue) compared to the active NECA–A2AR–mini-Gs structure (a–e) or ‘intermediate’ NECA–A2AR structure (orange) (f–j). d, i, Orthosteric binding site of the active A1R–Gi2 complex with ADO (purple ball and sticks) or A2AR with NECA (orange ball and sticks). ‘Toggle switch’ residue W6.48 and residues within 4 Å of ADO are labelled and shown as sticks. N, O and S atoms are coloured in blue, red and yellow, respectively. Dashed lines represent hydrogen bonds. e, j, Conserved class A GPCR motifs important for receptor activation (DRY motif, purple; NPXXY motif, blue; PIF motif, green).
Extended Data Fig. 7 Alignments of A1R with β2AR or dominant-negative Gαi2 with Gαs.
a, A1R alignment with β2AR. b, Gαs alignment with dominant-negative Gαi2. Key Ballesteros–Weinstein numbers are shown in red. Grey bars indicate the positions of the α-helices in the A1R–Gαi2 structure, whereas red bars indicate these regions in the β2AR–Gαs structure. Dominant-negative Gαi2 point mutations are highlighted in yellow.
Extended Data Fig. 8 Comparison of the A1R–Gi2 with β2AR–Gs structures.
Overlay of A1R–Gi2 with β2AR–Gs (PDB code 3SN6) complexes. (A1R–Gi2 is coloured as in Fig. 1; β2AR is in green, Gαs is in gold, Gβ is in light cyan, Gγ is in light purple).
Extended Data Fig. 9 View of key residues at the interface of A1R and Gβ, and Gα conformations.
a, A1R is in blue and Gβ is in dark cyan. b, c, Different views comparing DNGαi2 and Gαs from A1R and β2AR (PDB code 3SN6) receptor-bound structures (DNGαi2, pink; Gαs, gold). Spheres indicate the positions of the dominant-negative mutations on the DNGαi2 with N, O and C atoms coloured in blue, red and pink, respectively. The α5-helix bend and loops that are the most different between Gαi2 and Gαs are indicated.
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Draper-Joyce, C.J., Khoshouei, M., Thal, D.M. et al. Structure of the adenosine-bound human adenosine A1 receptor–Gi complex. Nature 558, 559–563 (2018). https://doi.org/10.1038/s41586-018-0236-6
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DOI: https://doi.org/10.1038/s41586-018-0236-6
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