In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.

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  1. 1.

    et al. International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol. Rev. 58, 281–341 (2006)

  2. 2.

    P2 receptors, platelet function and pharmacological implications. Thromb. Haemost. 99, 466–472 (2008)

  3. 3.

    , , & Pharmacochemistry of the platelet purinergic receptors. Purinergic Signal. 7, 305–324 (2011)

  4. 4.

    & Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc. Natl Acad. Sci. USA 95, 8070–8074 (1998)

  5. 5.

    et al. Major contribution of the P2Y1 receptor in purinergic regulation of TNFalpha-induced vascular inflammation. Circulation 123, 2404–2413 (2011)

  6. 6.

    , & P2Y1 purinoceptor-mediated Ca2+ signaling and Ca2+ wave propagation in dorsal spinal cord astrocytes. J. Neurosci. 20, 2800–2808 (2000)

  7. 7.

    , , & Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors. J. Neurosci. 23, 2348–2356 (2003)

  8. 8.

    et al. 2-Substitution of adenine nucleotide analogues containing a bicyclo[3.1.0]hexane ring system locked in a northern conformation: enhanced potency as P2Y1 receptor antagonists. J. Med. Chem. 46, 4974–4987 (2003)

  9. 9.

    et al. MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate], a potent, selective, and stable antagonist of the platelet P2Y1 receptor with strong antithrombotic activity in mice. J. Pharmacol. Exp. Ther. 316, 556–563 (2006)

  10. 10.

    et al. Discovery of 2-(phenoxypyridine)-3-phenylureas as small molecule P2Y1 antagonists. J. Med. Chem. 56, 1704–1714 (2013)

  11. 11.

    et al. High-resolution crystal structure of human protease-activated receptor 1. Nature 492, 387–392 (2012)

  12. 12.

    et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012)

  13. 13.

    et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010)

  14. 14.

    et al. Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 341, 1387–1390 (2013)

  15. 15.

    et al. Structure of the human kappa-opioid receptor in complex with JDTic. Nature 485, 327–332 (2012)

  16. 16.

    & Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995)

  17. 17.

    et al. Agonist-bound structure of the human P2Y12 receptor. Nature 509, 119–122 (2014)

  18. 18.

    et al. Structure of the human P2Y12 receptor in complex with an antithrombotic drug. Nature 509, 115–118 (2014)

  19. 19.

    , , & Architecture of P2Y nucleotide receptors: structural comparison based on sequence analysis, mutagenesis, and homology modeling. J. Med. Chem. 47, 5393–5404 (2004)

  20. 20.

    et al. Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites. J. Med. Chem. 41, 1456–1466 (1998)

  21. 21.

    , , , & Evidence for the recognition of non-nucleotide antagonists within the transmembrane domains of the human P2Y1 receptor. Drug Dev. Res. 57, 173–181 (2002)

  22. 22.

    et al. Conformationally constrained ortho-anilino diaryl ureas: discovery of 1-(2-(1′-neopentylspiro[indoline-3,4′-piperidine]-1-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea, a potent, selective, and bioavailable P2Y1 antagonist. J. Med. Chem. 56, 9275–9295 (2013)

  23. 23.

    et al. Discovery of 4-aryl-7-hydroxyindoline-based P2Y1 antagonists as novel antiplatelet agents. J. Med. Chem. 57, 6150–6164 (2014)

  24. 24.

    et al. Discovery of diarylurea P2Y1 antagonists with improved aqueous solubility. Bioorg. Med. Chem. Lett. 23, 3239–3243 (2013)

  25. 25.

    et al. A ligand channel through the G protein coupled receptor opsin. PLoS ONE 4, e4382 (2009)

  26. 26.

    et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012)

  27. 27.

    et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014)

  28. 28.

    , , , & Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008)

  29. 29.

    et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469, 175–180 (2011)

  30. 30.

    et al. Structure of an agonist-bound human A2A adenosine receptor. Science 332, 322–327 (2011)

  31. 31.

    et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012)

  32. 32.

    & Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)

  33. 33.

    Xds. Acta Crystallogr. D 66, 125–132 (2010)

  34. 34.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  35. 35.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  36. 36.

    et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

  37. 37.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

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This work was supported by the National Basic Research Program of China grants 2012CB518000, 2014CB910400 and 2012CB910400 (Q.Z., B.W.), CAS Strategic Priority Research Program XDB08020300 (B.W.), the National Science Foundation of China grants 31422017 (B.W.), 31370729 (Q.Z.) and 91313000 (H.J.), the National Science and Technology Major Project 2013ZX09507001 (H.J., Q.Z., B.W.), NIDDK, NIH Intramural Research Program grant Z01 DK031116-26 (K.A.J.), and the National Institutes of Health grant U54 GM094618 (V.C., V.K., R.C.S.). The authors thank A. Walker for assistance with manuscript preparation and S. M. Moss for technical assistance. The synchrotron radiation experiments were performed at the BL41XU of Spring-8 with approval of the Japan Synchrotron Radiation Research Institute (JASRI) (proposal no. 2014A1094 and 2014B1056). We thank the beamline staff members of the BL41XU for help with X-ray data collection.

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  1. CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China

    • Dandan Zhang
    • , Kaihua Zhang
    • , Jiang Wang
    • , Cuiying Yi
    • , Limin Ma
    • , Wenru Zhang
    • , Hong Liu
    • , Qiang Zhao
    •  & Beili Wu
  2. Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Zhan-Guo Gao
    • , Evgeny Kiselev
    • , Steven Crane
    • , Silvia Paoletta
    •  & Kenneth A. Jacobson
  3. Bridge Institute, Department of Chemistry, University of Southern California, Los Angeles, California 90089, USA

    • Gye Won Han
    • , Vadim Cherezov
    •  & Raymond C. Stevens
  4. Bridge Institute, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, USA

    • Vsevolod Katritch
    •  & Raymond C. Stevens
  5. Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China

    • Hualiang Jiang
  6. iHuman Institute, ShanghaiTech University, 99 Haike Road, Pudong, Shanghai 201203, China

    • Raymond C. Stevens


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D.Z. optimized the construct, developed the purification procedure and purified the P2Y1R proteins for crystallization, performed crystallization trials and optimized crystallization conditions. Z.-G.G. designed, performed and analysed ligand binding and competition assays of wild-type and mutant P2Y1R. K.Z. helped with construct and crystal optimization, and collected diffraction data. E.K. and J.W. helped with ligand synthesis of P2Y1R. S.C. performed and analysed ligand-binding assays. S.P. performed and analysed docking assays. C.Y. and L.M. expressed the P2Y1R proteins. W.Z. developed the initial expression and purification protocol for P2Y1R. G.W.H. helped to analyse the structures. H.L. oversaw ligand synthesis of P2Y1R. V.C. and V.K. helped to analyse the structures and assisted with manuscript preparation. H.J. and R.C.S. oversaw structure analysis/interpretation of P2Y1R. K.A.J. oversaw, designed and analysed ligand-binding assays, oversaw ligand synthesis, and assisted with manuscript preparation. Q.Z. and B.W. initiated the project, planned and analysed experiments, solved the structures, supervised the research and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Qiang Zhao or Beili Wu.

Atomic coordinates and structure factors for the P2Y1R–MRS2500 and P2Y1R–BPTU structures have been deposited in the Protein Data Bank with identification codes 4XNW and 4XNV.

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