Abstract

Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR–SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors.

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Acknowledgements

This work was supported by the National Key R&D Program of China, 2018YFA0507000; CAS Strategic Priority Research Program grant XDB08020000 (B.W., X.C.Z., Z.R. and X.L.); the Key Research Program of Frontier Sciences, CAS, grants QYZDB-SSW-SMC024 (B.W.) and QYZDB-SSW-SMC054 (Q.Z.); the National Science Foundation of China, grants 81525024 (Q.Z.) and 31301163 (C.X.); and the Program of Introducing Talents of Discipline to the Universities of the Ministry of Education (grant B08029) (J.L.). The authors thank M. Hanson, V. Cherezov and V. Katritch for careful review and scientific feedback on the manuscript; H. Zhang for guidance in handling radiolabeled chemicals; and J. W. Chin (Medical Research Council Laboratory of Molecular Biology, Cambridge) for providing plasmids (U6-PylT*)4/EF1α-PylRS, (U6-PylT*)4/EF1α-sfGFP(TAG) and peRF1 (E55D). The synchrotron radiation experiments were performed at BL41XU of SPring-8 with approval from the Japan Synchrotron Radiation Research Institute (proposal nos. 2014B1057, 2015A1026, 2015A1027, 2015B2026 and 2015B2027). We thank the BL41XU beamline staff members K. Hasegawa, H. Okumura and H. Murakami for help with X-ray data collection.

Author information

Author notes

  1. These authors contributed equally: Can Cao, Qiuxiang Tan.

Affiliations

  1. National Laboratory of Biomacromolecules, National Center of Protein Science–Beijing, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    • Can Cao
    • , Lingli He
    • , Ye Zhou
    • , Xianping Wang
    • , Xuemei Li
    • , Zihe Rao
    • , Yongfang Zhao
    •  & Xuejun C. Zhang
  2. CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    • Qiuxiang Tan
    • , Cuiying Yi
    • , Hualiang Jiang
    • , Qiang Zhao
    •  & Beili Wu
  3. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    • Qiuxiang Tan
    • , Anna Qiao
    • , Minmin Lu
    • , Hualiang Jiang
    •  & Qiang Zhao
  4. University of Chinese Academy of Sciences, Beijing, China

    • Can Cao
    • , Qiuxiang Tan
    • , Ye Zhou
    • , Anna Qiao
    • , Minmin Lu
    • , Qiang Zhao
    • , Xuejun C. Zhang
    •  & Beili Wu
  5. College of Life Science and Technology, Collaborative Innovation Center for Genetics and Development, and Key Laboratory of Molecular Biophysics of the Ministry of Education, Huazhong University of Science and Technology, Wuhan, China

    • Chanjuan Xu
    • , Yiwei Zhou
    •  & Jianfeng Liu
  6. Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China

    • Linlin Yang
  7. Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA, USA

    • Gye Won Han
  8. Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    • Huaiyu Yang
    •  & Hualiang Jiang
  9. iHuman Institute, ShanghaiTech University, Shanghai, China

    • Raymond C. Stevens
  10. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    • Raymond C. Stevens
    •  & Beili Wu
  11. CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences, Beijing, China

    • Qiang Zhao
    •  & Beili Wu

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Contributions

C.C. and Q.T. optimized the construct; developed the purification procedure and purified the PAFR proteins for crystallization; and performed crystallization trials and optimized crystallization conditions. C.X. and Yiwei Zhou designed, performed and analyzed Ca2+-flux and IP-accumulation assays of WT and mutant PAFRs. L.H. and C.C. designed, performed and analyzed smFRET assays. L.Y. performed and analyzed MD simulations and docking assays. C.C. and Ye Zhou designed, performed and analyzed ligand-binding assays of WT and mutant PAFRs. A.Q. and M.L. assisted in construct and crystal optimization. C.Y. expressed the PAFR proteins. G.W.H. assisted in structure refinement. X.W. and X.L. helped to develop the initial expression and purification protocol for PAFR. H.Y. oversaw computational assays. Z.R. oversaw structure analysis and interpretation. H.J. oversaw computational assays and structure analysis and interpretation. Y. Zhao oversaw smFRET assays. J.L. oversaw Ca2+-flux and IP-accumulation assays, and edited the manuscript. R.C.S. assisted in structure analysis and interpretation, and edited the manuscript. Q.Z. oversaw construct design, collected crystal diffraction data, solved the PAFR structures and assisted with manuscript preparation. X.C.Z. and B.W. initiated the project, planned and analyzed experiments, solved the structures, supervised the research and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Xuejun C. Zhang or Beili Wu.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Note

  2. Reporting Summary

  3. Source data for Fig. 4a-e

  4. Source data for Fig. 5

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https://doi.org/10.1038/s41594-018-0068-y