Abstract

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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Acknowledgements

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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Affiliations

  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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Contributions

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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https://doi.org/10.1038/nature14287

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