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Agonist-bound structure of the human P2Y12 receptor

Abstract

The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y12R provided useful insights into ligand binding1,2,3,4, the agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here we report the structures of the human P2Y12R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. These structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing questions surrounding P2Y12R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.

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Figure 1: Structure of the P2Y12R–2MeSADP and P2Y12R–2MeSATP complexes.
Figure 2: Comparison of the P2Y12R–2MeSADP (agonist) and P2Y12R–AZD1283 (antagonist) complexes.
Figure 3: P2Y12R ligand binding pocket for 2MeSADP.
Figure 4: Schematic illustration of conformational changes in P2Y12R extracellular region.

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Acknowledgements

This work was supported by the National Basic Research Program of China grants 2012CB910400, 2012CB518000 and 2014CB910400 (B.W., Q.Z.), the National Institutes of Health grants R01 AI100604 (B.W., Q.Z.) and U54 GM094618 (V.C., V.K., R.C.S.; Target GPCR-87), the National Science Foundation of China grants 31370729 and National Science and Technology Major Project 2013ZX09507001 and 2012ZX09301001 (B.W., Q.Z.), National Institutes of Health NIDDK Intramural Research Program (K.A.J.) and the National Natural Science Foundation of China 91313000 (H.J.). The authors thank S. Nylander, E. Kiselev and S. Moss for scientific feedback on the manuscript, A. Walker for assistance with manuscript preparation and K. Kadyshevskaya for help with figure preparation. The synchrotron radiation experiments were performed at the BL41XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal no. 2013B1049). We thank the beamline staff members of the BL41XU for help with X-ray data collection.

Author information

Authors and Affiliations

Authors

Contributions

J.Z. optimized the construct, expressed and purified human P2Y12R–BRIL for crystallization, developed the purification procedure, performed crystallization trials and optimized crystallization conditions. K.Z. helped with construct and crystal optimization and collected diffraction data. Z.-G.G. designed, performed and analysed ligand binding and competition assays of wild-type and mutant P2Y12R. S.P. performed and analysed docking assays. D.Z. helped in expression and purification. G.W.H. solved and refined the structure. T.L. helped the expression for crystallization trials. L.M. helped the expression for the activity assays. W.Z. developed the initial expression and purification protocol for P2Y12R. C.E.M. helped to design and analyse pharmacological experiments and wrote the manuscript. H.Y. helped to design and analyse docking assays. H.J. oversaw design and validation of P2Y12R models. V.C. helped to design and optimize LCP crystallization trials and processed crystallographic data and wrote the manuscript. V.K. performed and analysed molecular modelling simulations and wrote the manuscript. K.A.J. oversaw, designed and analysed ligand binding assays and docking and wrote the manuscript. R.C.S. oversaw expression, purification and crystallization, and structure analysis and interpretation of P2Y12R. B.W. and Q.Z. initiated the project, planned and analysed experiments, supervised the research and wrote the manuscript.

Corresponding authors

Correspondence to Beili Wu or Qiang Zhao.

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Competing interests

The authors declare no competing financial interests.

Additional information

Atomic coordinates and structure factors for the P2Y12R–2MeSADP and P2Y12R–2MeSATP structures have been deposited in the Protein Data Bank with identification codes 4PXZ and 4PY0, respectively.

Extended data figures and tables

Extended Data Figure 1 Crystals and electron density of nucleotides for P2Y12R–2MeSADP and P2Y12R–2MeSATP complexes.

a, Crystals of the P2Y12R–2MeSADP complex. The size of the crystals is roughly 80 × 50 × 5 μm. b, Crystals of the P2Y12R–2MeSATP complex. The size of the crystals is roughly 30 × 30 × 5μm. c, The 2mFo–DFc map for the 2MeSADP contoured at 1σ. d, The 2mFo–DFc map for the P2Y12R–2MeSATP contoured at 1σ. The relatively high B-factor of the γ-phosphate group (98 Å2) compared with β-phosphate and surrounding protein atoms (75 Å2), and the propensity of 2MeSATP to hydrolyse to 2MeSADP suggest partial occupancy for the γ-phosphate group. However, given the differences in crystal forms and packing, as well as the clear density of the γ-phosphate group, the P2Y12R–2MeSATP complex structure should provide relevant information about 2MeSATP binding.

Extended Data Figure 2 Crystal packing of P2Y12R–2MeSADP, P2Y12R–2MeSATP and P2Y12R–AZD1283 complexes.

a, Overall structure of the P2Y12R–2MeSADP complex, P2Y12R and BRIL are shown in cyan and blue, respectively. b, c, Crystal packing of P2Y12R–2MeSADP complex shown in two different views. d, Overall structure of the P2Y12R–2MeSATP complex, P2Y12R is shown in pink. e, f, Crystal packing of P2Y12R–2MeSATP complex in two different views. g, Overall structure of the P2Y12R–AZD1283 complex, P2Y12R is shown in orange. h, i, Crystal packing of P2Y12R–AZD1283 complex shown in two different views.

Extended Data Figure 3 Comparison of antagonist- (orange) and agonist- (green/cyan) bound P2Y12R structures with the PAR1 structure (yellow).

a, Side view of the three structures. The receptor structures are shown as cylindrical helices and AZD1283 and 2MeSADP are shown as sticks with green carbons and wheat carbons, respectively. b, Comparison view from the extracellular side. c, Comparison view from the intracellular side.

Extended Data Figure 4 The distortion of helix III by the disulphide bond.

a, Comparison of P2Y12R–AZD1283 (orange) and P2Y12R–2MeSADP (green/cyan). b, Corresponding positions of residues around C973.25 in P2Y12R–AZD1283 (orange) and P2Y12R–2MeSADP (green/cyan).

Extended Data Figure 5 Comparison of pocket 1 (a–c) and pocket 2 (d–f) of P2Y12R structures with different ligands.

a, d, The P2Y12R–2MeSADP structure. b, e, The P2Y12R–2MeSATP structure. c, f, The P2Y12R–AZD1283 structure. The 2MeSADP, 2MeSATP and AZD1283 ligands are shown in sticks with wheat, grey and green carbons, respectively.

Extended Data Figure 6 Functional properties of different ligands at P2Y12R.

Data (mean ± s.e.m.) were determined in triplicate. a, Parallel right shifts induced by antagonist AZD1283 (AZD) of the activation curves by agonist 2MeSADP in inhibition of cAMP production in P2Y12R expressing CHO cells. b, Parallel right shifts induced by antagonist ticagrelor (Tica) of the activation curves by agonist 2MeSADP in inhibition of cAMP production in P2Y12R expressing CHO cells. The pKb values of AZD and Tica are 8.17 ± 0.45 and 7.70 ± 0.18, respectively. c, Partial agonist effects of AR-C66096 (ARC) in inhibition of cAMP production in P2Y12R expressing CHO cells. The half-maximum effective concentration (EC50) value of AR-C66096 was determined to be 34.9 ± 2.9 nM, and its Emax 41.9 ± 3.6% compared with 2MeSADP as 100%. A final concentration of 10 μM forskolin was used in the experiment. DMSO was used as a solvent for the stock solution of forskolin, AZD1283 and ticagrelor. The stock solution of AR-C66096 was made with water.

Extended Data Figure 7 Docking models of different nucleotide analogues to the P2Y12R structure.

a, The crystal structure of P2Y12R–2MeSADP complex. b, Docking of 2MeSADP to the P2Y12R structure. c, Docking of ADP to the P2Y12R structure. d, The crystal structure of P2Y12R–2MeSATP complex. e, Docking of 2MeSATP to the P2Y12R structure. f, Docking of ATP to the P2Y12R structure. g, Docking of AR-C66096 to the P2Y12R structure. h, Docking of AR-C67085 to the P2Y12R structure. i, Docking of AR-C69931MX (cangrelor) to the P2Y12R structure. 2MeSADP and 2MeSATP poses from corresponding crystal structures are shown in stick with orange and grey carbons, respectively. Docking was performed to the conformation of P2Y12R found in the 2MeSADP-bound structure, and the docked ligands are shown in sticks with purple carbons. AR-C66096 and AR-C67085 show the same interactions observed in the 2MeSADP complex. In addition, the C2-propylthio substituent of AR-C66096 and AR-C67085 is located in a hydrophobic pocket in proximity to helix IV surrounded by F1063.34, Y1093.37, M1524.53 and L1554.56. The γ-phosphonate group is directed towards helix III and interacts with K802.60 and R933.21. The C2 substituent and the γ-phosphonate group of AR-C69931MX show similar orientation as observed in the docking pose of AR-C66096 and AR-C67085. The N6 substituent is directed towards helix VI in proximity to Y1093.37, Q1955.44, F2526.51, H2536.52 and R256 6.55.

Extended Data Figure 8 Ligands used in the docking studies.

The chemical structures of parts of ligands that are discussed and used in the docking studies are shown. Ticagrelor and AR-C78511 could not be docked in a conformation similar to 2MeSADP because the presence of their bulky N6substituents would cause a steric clash with helices V and VI. AR-C78511 was previously shown to lack partial agonist properties.

Extended Data Table 1 Data collection and refinement statistics
Extended Data Table 2 Binding affinities for different P2Y12R constructs

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Zhang, J., Zhang, K., Gao, ZG. et al. Agonist-bound structure of the human P2Y12 receptor. Nature 509, 119–122 (2014). https://doi.org/10.1038/nature13288

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