Abstract

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin–arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a 20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

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Protein Data Bank

Data deposits

The coordinates of the rhodopsin–arrestin complex and diffraction data have been deposited in the Protein Data Bank under accession number 4ZWJ.

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Acknowledgements

Portions of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. Use of the LCLS at the SLAC National Accelerator Laboratory is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. Parts of the sample injector used at LCLS for this research was funded by the National Institutes of Health, P41GM103393, formerly P41RR001209. We thank staff members of the Life Science Collaborative Access Team (ID-21) of the Advanced Photon Source (APS) for assistance in data collection at the beam lines of sector 21, which is in part funded by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817), and the General Medicine Collaborative Access Team for assistance in data collection at the beam lines of sector 23 (ID-23), funded in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). Use of APS was supported by the Office of Science of the US Department of Energy, under contract no. DE-AC02-06CH11357. This work was supported in part by the Jay and Betty Van Andel Foundation, Ministry of Science and Technology (China) grants 2012ZX09301001 and 2012CB910403, 2013CB910600, XDB08020303, 2013ZX09507001, Amway (China), National Institute of Health grants, DK071662 (H.E.X.); GM073197 and GM103310 (C.S.P. and B.C.); GM102545 and GM104212 (K.M.); EY011500 and GM077561 (V.V.G.), EY005216 and P30 EY000331 (W.L.H.), the National Institutes of Health Common Fund in Structural Biology grants P50 GM073197 (V.C. and R.C.S.), P50 GM073210 (M.C.), and GM095583 (P.F.); National Institute of General Medical Sciences PSI: Biology grants U54 GM094618 (V.C., V.K., and R.C.S.), GM108635 (V.C.), U54 GM094599 (P.F.), GM097463 (J.S.), and U54 GM094586 (JCSG); NSF Science and Technology Center award 1231306 (J.C.H.S., P.F. and U.W.); Swiss National Science Foundation grant 31003A_141235 (J.S.); the Canada Excellence Research Chair program and the Anne & Max Tanenbaum Chair in Neuroscience at the University of Toronto (O.P.E.); and Science Foundation Ireland, grant 12/IA/1255 (M.C.). Parts of this work were also supported by the Helmholtz Gemeinschaft, the DFG Cluster of Excellence Center for Ultrafast Imaging, and the BMBF project FKZ 05K12CH1 (H.N.C., A.B., C.G., O.Y., T.W.); the Irene and Eric Simon Brain Research Foundation (R.L.). We thank A. Brunger and O. Zeldin for analysing the XFEL data and for advising on refinement; B. Weis for advice on twin refinement and structure validation; J. Rini for advice on the piggyBac expression system; A. Lebedev for his advice regarding the Zanuda program and the choice of the space group; and A. Walker for final editing of the manuscript. C.G. kindly thanks the PIER Helmholtz-Graduate School and the Helmholtz Association for financial support. We also thank the TianHe research and development team of National University of Defense Technology (NUDT) for computational resources.

Author information

Author notes

    • Yanyong Kang
    • , X. Edward Zhou
    • , Xiang Gao
    •  & Yuanzheng He

    These authors contributed equally to this work.

    • Ned Van Eps

    Present address: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Affiliations

  1. Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA

    • Yanyong Kang
    • , X. Edward Zhou
    • , Xiang Gao
    • , Yuanzheng He
    • , Parker W. de Waal
    • , Jiyuan Ke
    • , M. H. Eileen Tan
    • , Chenghai Zhang
    • , Kelly M. Suino-Powell
    • , Xin Gu
    • , Kuntal Pal
    • , Jinming Ma
    • , Xiaoyong Zhi
    • , Karsten Melcher
    •  & H. Eric Xu
  2. Department of Chemistry and Biochemistry, and Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, Arizona 85287-1604, USA

    • Wei Liu
    • , Nadia A. Zatsepin
    • , Dingjie Wang
    • , Daniel James
    • , Shibom Basu
    • , Shatabdi Roy-Chowdhury
    • , Chelsie E. Conrad
    • , Jesse Coe
    • , Haiguang Liu
    • , Stella Lisova
    • , Christopher Kupitz
    • , Ingo Grotjohann
    • , Raimund Fromme
    • , John C. H. Spence
    • , Petra Fromme
    •  & Uwe Weierstall
  3. Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, California 90089, USA

    • Andrii Ishchenko
    • , Gye Won Han
    • , Raymond C. Stevens
    •  & Vadim Cherezov
  4. Center for Free Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany

    • Anton Barty
    • , Thomas A. White
    • , Oleksandr Yefanov
    • , Cornelius Gati
    •  & Henry N. Chapman
  5. Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • Qingping Xu
  6. Department of Obstetrics & Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    • M. H. Eileen Tan
    •  & Jun Li
  7. The National Resource for Automated Molecular Microscopy, New York Structural Biology Center, New York, New York 10027, USA

    • Arne Moeller
    • , Clinton S. Potter
    •  & Bridget Carragher
  8. Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, USA

    • Graham M. West
    • , Bruce D. Pascal
    •  & Patrick R. Griffin
  9. Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA

    • Ned Van Eps
    •  & Wayne L. Hubbell
  10. Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Lydia N. Caro
    •  & Oliver P. Ernst
  11. Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA

    • Sergey A. Vishnivetskiy
    • , Regina J. Lee
    •  & Vsevolod V. Gurevich
  12. Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • Sébastien Boutet
    • , Garth J. Williams
    •  & Marc Messerschmidt
  13. BioXFEL, NSF Science and Technology Center, 700 Ellicott Street, Buffalo, New York 14203, USA

    • Marc Messerschmidt
    •  & Yingming Zhao
  14. Department of Physics, Arizona State University, Tempe, Arizona 85287, USA

    • Nadia A. Zatsepin
    • , Dingjie Wang
    • , Daniel James
    • , Shibom Basu
    • , Shatabdi Roy-Chowdhury
    • , John C. H. Spence
    •  & Uwe Weierstall
  15. Beijing Computational Science Research Center, Haidian District, Beijing 10084, China

    • Haiguang Liu
  16. Department of Physics, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, USA

    • Christopher Kupitz
  17. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    • Yi Jiang
    • , Minjia Tan
    • , Huaiyu Yang
    •  & Hualiang Jiang
  18. Swiss Light Source at Paul Scherrer Institute, CH-5232 Villigen, Switzerland

    • Meitian Wang
  19. Department of Biological Sciences, Bridge Institute, University of Southern California, Los Angeles, California 90089, USA

    • Zhong Zheng
    • , Vsevolod Katritch
    •  & Raymond C. Stevens
  20. School of Medicine and School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland

    • Dianfan Li
    • , Nicole Howe
    •  & Martin Caffrey
  21. Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois 60637, USA

    • Yingming Zhao
  22. Laboratory of Biomolecular Research at Paul Scherrer Institute, CH-5232 Villigen, Switzerland

    • Jörg Standfuss
  23. Department of Biology, Universität Konstanz, 78457 Konstanz, Germany

    • Kay Diederichs
  24. Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

    • Yuhui Dong
  25. Centre for Ultrafast Imaging, 22761 Hamburg, Germany

    • Henry N. Chapman
  26. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Oliver P. Ernst
  27. iHuman Institute, ShanghaiTech University, 2F Building 6, 99 Haike Road, Pudong New District, Shanghai 201210, China

    • Raymond C. Stevens
  28. VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    • H. Eric Xu

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Contributions

Y.K. initiated the project, developed the expression and purification methods for rhodopsin–arrestin complex, and bulk-purified expression constructs and proteins used in LCP crystallization for the SFX method; X.E.Z. collected the synchrotron data, helped with the SFX data collection, processed the data, and solved the structures; X. Gao expressed and purified rhodopsin–arrestin complexes, characterized their binding and thermal stability, discovered the initial crystallization conditions with 9.7 MAG (1-(9Z-hexadecenoyl)-rac-glycerol), prepared most crystals for synchrotron data collection, prepared all crystals for the final data collection by SFX, helped with SFX data collection, and established the initial cross-linking method for the rhodopsin–arrestin complex; Y.H. designed and performed Tango assays and disulfide bond cross-linking experiments; C.Z. developed the mammalian expression methods; P.W.d.W. helped with XFEL data processing and performed computational experiments; J.K., M.H.E.T., K.M.S.-P., K.P., J.M., Y.J., X.Z., and X. Gu performed cell culture, mutagenesis, protein purification, rhodopsin–arrestin binding experiments; W.L. and A.I. grew crystals and collected synchrotron data at APS and SFX data at LCLS, G.W.H. and Q.X. determined and validated the structure. Z.Z. and V.K. constructed the full model, the phosphorylated rhodopsin–arrestin model, and helped writing the paper; D.W., S.L., D.J., C.K., Sh.B., and N.A.Z. helped with XFEL data collection and initial data analysis; Sé.B., M.M., and G.J.W. set up the XFEL experiment, performed the data collection, and commented on the paper. A.B., T.A.W., C.G., O.Y., and H.N.C. helped with XFEL data collection and data analysis, processed the data and helped with structure validation. G.M. W., B.D.P., and P.R.G. performed HDX experiments and helped with manuscript writing. J.L. helped initiate this collaborative project and with writing the paper. M.W. collected the 7.7 Å dataset at the Swiss Light Source. A.M., C.S.P., and B.C. were responsible for electron microscopy images of rhodopsin–arrestin complexes. M.T. and Y.Z. performed mass spectrometry experiments to validate the protein contents in the crystals; D.L., N. H., and M.C. provided the 9.7 MAG phase diagram and helped with SFX data collection and with writing the paper. J.S. provided a computational model of the rhodopsin–arrestin complex and helped with discussion and writing; K.D., H.L., and Y.D. helped with data analysis and twinning problems; R.J.L. constructed single-Cys arrestin-1 mutants for DEER and tested their binding to rhodopsin; S.A.V. expressed these mutants in Escherichia coli and purified them; V.V.G. provided arrestin genes, designed single-Cys arrestin-1 mutants for DEER, and helped analysing the data and writing the paper. H.Y. and H.J. performed computational modelling, figure preparation, and helped with writing the paper; J.C.H.S. and U.W. designed the LCP injector and helped with data collection. Sh.B., S.R.-C., C.E.C., J.C., C.K., I.G., P.F., and R.F. helped with data collection, on-site crystal characterization as well as data analysis, and validation of the structure. L.N.C. and O.P.E. generated the Y74C/C140S/C316S stable cell line, characterized and provided the rhodopsin mutant sample for DEER measurements. N.V.E. and W.L.H. incorporated rhodopsin into nanodiscs, spin-labelled rhodopsin and arrestin, performed DEER experiments and helped with manuscript writing. R.C.S. supervised crystal growth, data collection, structure solution and validation, and helped with manuscript writing. V.C. was the Principal Investigator of the LCLS data collection, supervised crystal growth, data collection at APS and LCLS, structure solution and validation, and helped with manuscript writing; K.M. supervised research, analysed data, and helped with writing the paper. H.E.X. conceived the project, designed the research, performed synchrotron and LCLS data collection and structure solution, and wrote the paper with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to H. Eric Xu.

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https://doi.org/10.1038/nature14656

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