ARTICLE https://doi.org/10.1038/s41586-018-0236-6 Structure of the adenosine-bound human adenosine A 1 receptor–G i complex Christopher J. Draper-Joyce 1,6 , Maryam Khoshouei 2,3,6 , David M. Thal 1 , Yi-Lynn Liang 1 , Anh T. N. Nguyen 1 , Sebastian G. B. Furness 1 , Hariprasad Venugopal 4 , Jo-Anne Baltos 1 , Jürgen M. Plitzko 2 , Radostin Danev 2 , Wolfgang Baumeister 2 , Lauren T. May 1 , Denise Wootten 1,5 , Patrick M. Sexton 1,5 *, Alisa Glukhova 1 * & Arthur Christopoulos 1 * The class A adenosine A 1 receptor (A 1 R) is a G-protein-coupled receptor that preferentially couples to inhibitory G i/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 A 1 R in complex with adenosine and heterotrimeric G i2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A 1 R, 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 A 1 R primarily via amino acids in the C terminus of the Gα i α5-helix, concomitant with a 10.5 Å outward movement of the A 1 R transmembrane domain 6. Comparison with the agonist-bound β 2 adrenergic receptor– G s -protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A 1 R structure provides molecular insights into receptor and G-protein selectivity. Adenosine (ADO) receptors comprise four subtypes within the class A G-protein-coupled receptor (GPCR) superfamily that mediate the actions of the purine nucleoside, ADO 1 . Activation of the A 1 R is thera- peutically desirable for ischaemia-reperfusion injury, atrial fibrillation, neuropathic pain and others 1 . Although ADO is used clinically to treat supraventricular tachycardia, the development of A 1 R-selective agonists for a broader range of disorders has thus far failed, primarily owing to dose-limiting on-target adverse effects 2 . Alternative approaches are thus necessary for improved A 1 R drug action, with studies focusing on the potential for greater A 1 R selectivity through targeting allosteric sites, or via development of A 1 R conformational state-selective biased agonists that can promote beneficial signalling while sparing pathways mediating on-target adverse effects 3,4 . One area for the development of selective A 1 R-targeting drugs is the use of structure-based approaches that leverage advances in GPCR structural biology. Indeed, inactive-state, antagonist-bound, struc- tures of the A 1 R were solved using X-ray crystallography 5,6 . However, these studies required modification of the A 1 R via thermostabilizing mutations and/or fusion proteins, and cannot inform on mechanisms underlying agonist binding, A 1 R activation and G-protein interac- tion. These features are necessary for the rational design of selective A 1 R activators, biased agonists or positive allosteric modulators. An alternative approach to overcoming the current dearth of active-state, G-protein-bound, GPCRs is the use of single-particle cryo-electron microscopy (cryo-EM) 7–9 . The promise of cryo-EM in yielding active- state GPCR complexes has recently been realized through the solution of several receptor structures bound to agonists and the heterotrimeric G s protein, complementing the only crystal structure so far, to our knowledge, of an agonist-bound GPCR–G-protein complex, that of the β 2A R complexed to G s protein 7–10 . However, the A 1 R preferentially couples to the G i/o family of G-proteins 1 . Indeed, of the more than 800 human GPCRs, most preferentially couple to G i/o proteins. The G i/o family has four members, which are the most abundantly expressed G proteins throughout the body 11 . G i/o protein activation is typically associated with inhibition of adenylate cyclase, resulting in reduced cAMP accumulation, but they also regulate numerous effectors includ- ing enzymes, ion channels and small GTPases. Based predominantly on the high expression of both G i2 proteins and A 1 Rs in brain and, albeit to a lesser degree, in cardiac tissues (both major organs for A 1 R therapies), we chose to focus on G i2 as a transducer for the A 1 R. Here we report the first, to our knowledge, structure of a GPCR coupled to a heterotrimeric G i protein, specifically the A 1 R–G i2 complex bound to its endogenous agonist, ADO, solved using Volta phase-plate (VPP) cryo-EM. Solving the A 1 R–G i2 complex To facilitate complex formation, A 1 R and G i2 were expressed sepa- rately in HighFive insect cells and combined after solubilizing in lauryl maltose-neopentyl glycol (LMNG) and cholesteryl hemisuccinate (CHS) with addition of apyrase and ADO (Extended Data Fig. 1). Stabilization of the A 1 R–G i2 complex was achieved by introducing four Gα i2 subunit mutations that alter nucleotide binding and affinity for Gβγ 8 . This dominant-negative G i2 (DNG i2 ) was sufficient to enable formation of a stable interaction with the receptor while insensitive to GTP (Extended Data Figs. 1 and 2c). The antagonist dipropylcyclopen- tylxanthine (DPCPX) displayed similar affinities for the A 1 R whether alone or in the presence of wild-type Gα i2 or DNG i2 (Extended Data Fig. 2a), whereas ADO displayed biphasic binding curves with a sim- ilar dispersion of high and low affinity states in the presence, but not absence, of either wild-type Gα i2 or DNG i2 (Extended Data Fig. 2b); a characteristic feature of agonist binding to many GPCRs 12 . By con- trast, agonist-mediated [ 35 S]GTPγS binding to activated Gα subunits was only observed upon combination of the A 1 R with wild-type Gα i2 (Extended Data Fig. 2c). The A 1 R–G i2 complex in LMNG detergent micelles was visualized using a Titan Krios microscope equipped with a VPP. After imaging and initial 2D classification (Extended Data Fig. 3a, b), 3D classification yielded a final map at a nominal resolution of 3.6 Å reconstructed from 263,321 particle projections (Fig. 1, Extended Data Fig. 3c, Extended 1 Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia. 2 Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany. 3 Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland. 4 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. 5 School of Pharmacy, Fudan University, Shanghai, China. 6 These authors contributed equally: Christopher J. Draper-Joyce, Maryam Khoshouei. *e-mail: patrick.sexton@monash.edu; alisa.glukhova@monash.edu; arthur.christopoulos@monash.edu N AT U R E | www.nature.com/nature © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.