Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural basis for signal recognition and transduction by platelet-activating-factor receptor

Nature Structural & Molecular Biology volume 25, pages 488–495 (2018)Cite this article

Subjects

This article has been updated

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Structures of the PAFR–SR 27417 and PAFR–ABT-491 complexes.
Fig. 2: Ligand-binding modes of PAFR.
Fig. 3: Conformational changes of the helical bundle in PAFR.
Fig. 4: smFRET assay of PAFR, measuring the distance between the intracellular tips of helices II and IV.
Fig. 5: IP-accumulation assays of PAFR.
Fig. 6: Helix VIII in the PAFR–SR 27417 structure.

Similar content being viewed by others

Change history

  • 08 May 2019

    In the version of this article initially published, Supplementary Table 1 and the Supplementary Note were omitted from the Supplementary Text and Figures file. The error has been corrected.

References

  1. Hwang, S. B. Specific receptors of platelet-activating factor, receptor heterogeneity, and signal transduction mechanisms. J. Lipid Mediat. 2, 123–158 (1990).

    CAS  PubMed  Google Scholar 

  2. Braquet, P., Touqui, L., Shen, T. Y. & Vargaftig, B. B. Perspectives in platelet-activating factor research. Pharmacol. Rev. 39, 97–145 (1987).

    CAS  PubMed  Google Scholar 

  3. Chao, W. & Olson, M. S. Platelet-activating factor: receptors and signal transduction. Biochem. J. 292, 617–629 (1993).

    Article  CAS  Google Scholar 

  4. Dupré, D. J., Le Gouill, C., Rola-Pleszczynski, M. & Stanková, J. Inverse agonist activity of selected ligands of platelet-activating factor receptor. J. Pharmacol. Exp. Ther. 299, 358–365 (2001).

    PubMed  Google Scholar 

  5. Herbert, J. M. et al. Biochemical and pharmacological activities of SR 27417, a highly potent, long-acting platelet-activating factor receptor antagonist. J. Pharmacol. Exp. Ther. 259, 44–51 (1991).

    CAS  PubMed  Google Scholar 

  6. Albert, D. H. et al. Pharmacology of ABT-491, a highly potent platelet-activating factor receptor antagonist. Eur. J. Pharmacol. 325, 69–80 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Thorsen, T. S., Matt, R., Weis, W. I. & Kobilka, B. K. Modified T4 lysozyme fusion proteins facilitate G protein-coupled receptor crystallogenesis. Structure 22, 1657–1664 (2014).

    Article  CAS  Google Scholar 

  9. Ballesteros, J. & Weinstein, H. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Chrencik, J. E. et al. Crystal structure of antagonist bound human lysophosphatidic acid receptor 1. Cell 161, 1633–1643 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Hua, T. et al. Crystal structure of the human cannabinoid receptor CB1. Cell 167, 750–762.e14 (2016).

    Article  CAS  Google Scholar 

  16. Curtin, M. L. et al. Discovery and evaluation of a series of 3-acylindole imidazopyridine platelet-activating factor antagonists. J. Med. Chem. 41, 74–95 (1998).

    Article  CAS  Google Scholar 

  17. Ryan, S. D., Harris, C. S., Carswell, C. L., Baenziger, J. E. & Bennett, S. A. Heterogeneity in the sn-1 carbon chain of platelet-activating factor glycerophospholipids determines pro- or anti-apoptotic signaling in primary neurons. J. Lipid Res. 49, 2250–2258 (2008).

    Article  CAS  Google Scholar 

  18. Wess, J., Han, S. J., Kim, S. K., Jacobson, K. A. & Li, J. H. Conformational changes involved in G-protein-coupled-receptor activation. Trends Pharmacol. Sci. 29, 616–625 (2008).

    Article  CAS  Google Scholar 

  19. Kuwasako, K., Kitamura, K., Nagata, S., Hikosaka, T. & Kato, J. Structure-function analysis of helix 8 of human calcitonin receptor-like receptor within the adrenomedullin 1 receptor. Peptides 32, 144–149 (2011).

    Article  CAS  Google Scholar 

  20. Wacker, D., Stevens, R. C. & Roth, B. L. How ligands illuminate GPCR molecular pharmacology. Cell 170, 414–427 (2017).

    Article  CAS  Google Scholar 

  21. Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011).

    Article  CAS  Google Scholar 

  22. Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W. & Ernst, O. P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Zhang, D. et al. Two disparate ligand-binding sites in the human P2Y1 receptor. Nature 520, 317–321 (2015).

    Article  CAS  Google Scholar 

  25. Ernst, O. P. et al. Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits. J. Biol. Chem. 275, 1937–1943 (2000).

    Article  CAS  Google Scholar 

  26. Delos Santos, N. M., Gardner, L. A., White, S. W. & Bahouth, S. W. Characterization of the residues in helix 8 of the human β1-adrenergic receptor that are involved in coupling the receptor to G proteins. J. Biol. Chem. 281, 12896–12907 (2006).

    Article  Google Scholar 

  27. Sounier, R. et al. Propagation of conformational changes during μ-opioid receptor activation. Nature 524, 375–378 (2015).

    Article  CAS  Google Scholar 

  28. Liang, Y. L. et al. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118–123 (2017).

    Article  CAS  Google Scholar 

  29. Zhang, Y. et al. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248–253 (2017).

    Article  CAS  Google Scholar 

  30. Liang, Y. L. et al. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature 555, 121–125 (2018).

    Article  CAS  Google Scholar 

  31. Zhang, H. et al. Structural basis for selectivity and diversity in angiotensin II receptors. Nature 544, 327–332 (2017).

    Article  CAS  Google Scholar 

  32. Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011).

    Article  CAS  Google Scholar 

  33. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  34. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  38. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  39. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008).

    Article  CAS  Google Scholar 

  40. Klauda, J. B. et al. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. J. Phys. Chem. B 114, 7830–7843 (2010).

    Article  CAS  Google Scholar 

  41. Vanommeslaeghe, K. & MacKerell, A. D. Jr. Automation of the CHARMM general force field (CGenFF) I: bond perception and atom typing. J. Chem. Inf. Model. 52, 3144–3154 (2012).

    Article  CAS  Google Scholar 

  42. Vanommeslaeghe, K., Raman, E. P. & MacKerell, A. D. Jr. Automation of the CHARMM General Force Field (CGenFF) II: assignment of bonded parameters and partial atomic charges. J. Chem. Inf. Model. 52, 3155–3168 (2012).

    Article  CAS  Google Scholar 

  43. Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101 (2007).

    Article  Google Scholar 

  44. Berendsen, H. J. C., Postma, J. P. M., Vangunsteren, W. F., Dinola, A. & Haak, J. R. Molecular-dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690 (1984).

    Article  CAS  Google Scholar 

  45. Miyamoto, S. & Kollman, P. A. Settle: an analytical version of the shake and rattle algorithm for rigid water models. J. Comput. Chem. 13, 952–962 (1992).

    Article  CAS  Google Scholar 

  46. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: a linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997).

    Article  CAS  Google Scholar 

  47. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593 (1995).

    Article  CAS  Google Scholar 

  48. Chatterjee, A., Sun, S. B., Furman, J. L., Xiao, H. & Schultz, P. G. A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry 52, 1828–1837 (2013).

    Article  CAS  Google Scholar 

  49. Schmied, W. H., Elsässer, S. J., Uttamapinant, C. & Chin, J. W. Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J. Am. Chem. Soc. 136, 15577–15583 (2014).

    Article  CAS  Google Scholar 

  50. Juette, M. F. et al. Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale. Nat. Methods 13, 341–344 (2016).

    Article  Google Scholar 

Download references

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.

Authors and 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

Authors
  1. Can Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qiuxiang Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chanjuan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lingli He
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Linlin Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ye Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yiwei Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Anna Qiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Minmin Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Cuiying Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Gye Won Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Xianping Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Xuemei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Huaiyu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Zihe Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Hualiang Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Yongfang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Jianfeng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Raymond C. Stevens
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Qiang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Xuejun C. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Beili Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Xuejun C. Zhang or Beili Wu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

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

Reporting Summary

Source data for Fig. 4a-e

Source data for Fig. 5

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cao, C., Tan, Q., Xu, C. et al. Structural basis for signal recognition and transduction by platelet-activating-factor receptor. Nat Struct Mol Biol 25, 488–495 (2018). https://doi.org/10.1038/s41594-018-0068-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing