Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser (original) (raw)

Accession codes

Primary accessions

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.

References

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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

  1. Ned Van Eps
    Present address: †Present address: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.,
  2. Yanyong Kang, X. Edward Zhou, Xiang Gao and Yuanzheng He: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, 49503, Michigan, 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, 85287-1604, Arizona, 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, 90089, California, USA
    Andrii Ishchenko, Gye Won Han, Raymond C. Stevens & Vadim Cherezov
  4. Center for Free Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Hamburg, 22607, 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, 94025, California, 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, 10027, New York, USA
    Arne Moeller, Clinton S. Potter & Bridget Carragher
  8. Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, 33458, Florida, 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, 90095, California, USA
    Ned Van Eps & Wayne L. Hubbell
  10. Department of Biochemistry, University of Toronto, Toronto, M5S 1A8, Ontario, Canada
    Lydia N. Caro & Oliver P. Ernst
  11. Department of Pharmacology, Vanderbilt University, Nashville, 37232, Tennessee, USA
    Sergey A. Vishnivetskiy, Regina J. Lee & Vsevolod V. Gurevich
  12. Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, Menlo Park, 94025, California, USA
    Sébastien Boutet, Garth J. Williams & Marc Messerschmidt
  13. BioXFEL, NSF Science and Technology Center, 700 Ellicott Street, Buffalo, 14203, New York, USA
    Marc Messerschmidt & Yingming Zhao
  14. Department of Physics, Arizona State University, Tempe, 85287, Arizona, 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, 53211, Wisconsin, 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, Villigen, CH-5232, Switzerland
    Meitian Wang
  19. Department of Biological Sciences, Bridge Institute, University of Southern California, Los Angeles, 90089, California, 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, 60637, Illinois, USA
    Yingming Zhao
  22. Laboratory of Biomolecular Research at Paul Scherrer Institute, Villigen, CH-5232, Switzerland
    Jörg Standfuss
  23. Department of Biology, Universität Konstanz, Konstanz, 78457, 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, Hamburg, 22761, Germany
    Henry N. Chapman
  26. Department of Molecular Genetics, University of Toronto, Toronto, M5S 1A8, Ontario, 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

Authors

  1. Yanyong Kang
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  2. X. Edward Zhou
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  3. Xiang Gao
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  4. Yuanzheng He
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  13. Jiyuan Ke
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  23. Kelly M. Suino-Powell
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  24. Xin Gu
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  26. Jinming Ma
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  53. Jörg Standfuss
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  72. 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.

Corresponding author

Correspondence toH. Eric Xu.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Constitutively active rhodopsin interacts with arrestin and GαCT-HA.

a, SDS–PAGE of N-terminally MBP-tagged wild-type and mutant rhodopsin. b, Non-cropped versions of the pull-down assay gels shown in Fig. 1b. The interactions between mouse wild-type arrestin and human wild-type or E1133.28Q rhodopsin are very weak. In contrast, the interaction between constitutively active rhodopsin (E1133.28Q/M2576.40Y) and pre-activated L374A/V375A/F376A arrestin (3A arrestin) is strong and is further increased in the presence of 10 μM all-_trans-_retinal. Input: 5% of the binding reaction. Bottom panels show the rhodopsin loading controls. c, Schematic representation of the AlphaScreen assay. d, AlphaScreen binding assay between E1133.28Q/M2576.40Y rhodopsin and GαCT-HA (TGGRVLEDLKSCGLF) in the presence and absence of 5 µM all-_trans-_retinal. The two left columns show the controls with ‘peptide only’ and ‘rhodopsin only’. (n = 3, error bars, s.d.). e, Determination of the affinity of the interaction between rhodopsin E1133.28Q/M2576.40Y and GαCT-HA by homologous competition. His6–MBP–rhodopsin mutant protein was immobilized on Ni-acceptor beads and biotinylated GαCT-HA on streptavidin donor beads. Binding between rhodopsin and arrestin brings donor and acceptor beads into close proximity, resulting in the indicated binding signal. Non-biotinylated GαCT-HA competed for the interaction with an IC50 of ∼700 nM (n = 3, error bars, s.d.).

Extended Data Figure 2 Purification and crystallization of T4L–rhodopsin–arrestin.

a, Purification of the T4L–rhodopsin–arrestin (T4L–Rho–Arr) complex. His8–MBP–MBP–T4L–Rho–Arr complex was first purified by amylose column chromatography (lane 1). The His8–MBP–MBP tandem tag was then released by cleavage with 3C protease (lane 2) and removed by binding to Ni-NTA beads to recover pure T4L–rhodopsin–arrestin (T4L–Rho–Arr) protein (lane 3). b, Analytical gel filtration profile of the T4L–rhodopsin–arrestin complex. T4L–rhodopsin–arrestin eluted mostly as monomers with a small proportion of oligomers. The molecular weights of protein standards are indicated at the top. c, Thermal stability shift analysis of T4L–rhodopsin–arrestin. T4L–rhodopsin–arrestin is relatively stable with a _T_m of 59 °C. d, e, Crystals of T4L–rhodopsin–arrestin in lipid cubic phase under bright-field illumination (d) and polarized light (e). f, X-ray diffraction pattern of a T4L–rhodopsin–arrestin crystal recorded at LS-CAT of APS. The green ring indicates the position of reflections at 8.0 Å resolution.

Extended Data Figure 3 Electron density map for the overall complex and the key interfaces based on the XFEL data.

a, A 2F o − F c electron density map contoured at 1_σ_ of the arrestin finger loop, which forms the key interface with TM7 and helix 8. b, A 2F o − F c electron density map contoured at 1_σ_ of the loop between TM5 and TM6, which forms the key interface with the β-strand following the finger loop. c, A 3,000 K simulated annealing omit map (2F o − F c electron density map contoured at 1_σ_) calculated from the 3.8 Å/3.8 Å/3.3 Å XFEL data supports the overall arrangement of the rhodopsin–arrestin complex. In all panels, the complex structure is shown with rhodopsin coloured in green and arrestin in brown. d, The C-loop with a 2F o − F c composite omit map at 1_σ_ calculated from the 3.8 Å/3.8 Å/3.3 Å truncated XFEL data. Key residues are labelled.

Extended Data Figure 4 Structure similarity of the four rhodopsin–arrestin complexes in the asymmetric units and the interface between rhodopsin and arrestin.

a, Two 90° views of the superposition of the four rhodopsin–arrestin complexes are shown as cartoon representation. The four complexes have an r.m.s.d. of less than 0.5 Å in the Cα atoms of rhodopsin and arrestin. b, Close-up view of arrestin-binding sites in rhodopsin. The four arrestin-binding sites (P1–P4) are highlighted in brown on the rhodopsin surface. The rhodopsin C-terminal tail/arrestin interface (P4) is based on computational modelling and disulfide cross-linking data. c, Rhodopsin-binding sites in arrestin. The four rhodopsin-binding sites (P1–P4) are highlighted in green on the arrestin surface.

Extended Data Figure 5 Conformational modelling of the rhodopsin–arrestin full length complex.

a, An overview of the computational model. b, Predicted interactions of the rhodopsin C terminus with arrestin, showing strong to medium pairwise restraints between Cβ atoms of rhodopsin and arrestin residues identified by disulfide crosslinking. c, Same as in b, but showing predicted hydrogen bonding and ionic interactions for the C-terminal residues of rhodopsin.

Extended Data Figure 6 Dynamics of free 3A arrestin and rhodopsin-bound arrestin determined by HDX.

a, HDX perturbation map between rhodopsin-bound arrestin and free arrestin, which is derived from the difference in the HDX rate between rhodopsin-bound arrestin and free arrestin. The bars below the arrestin sequence represent the peptide fragments resolved by mass spectrometry and the colours of the bars indicate the relative decrease in deuterium exchange (colour code at bottom). b, The thermal stability of free 3A arrestin and the rhodopsin–arrestin complex shows that the rhodopsin–arrestin complex is more stable than free 3A arrestin.

Extended Data Figure 7 Cell-based Tango assays to validate the rhodopsin–arrestin interface.

a, Cartoon illustration of the Tango assay for rhodopsin–arrestin interactions in cells. b, c, Mutations of key arrestin (b) and rhodopsin (c) residues that mediate the rhodopsin–arrestin interactions. Tango assay were performed in the absence or presence of 10 µM all-_trans_-retinal (ATR). (n = 3, error bars, s.d.).

Extended Data Figure 8 Control experiments for disulfide bond cross-linking specificity.

a, The product of the cross-linking reaction of finger loop residue G77C with N3107.57C of TM7 was confirmed by western blots using anti-Flag antibody (which detects arrestin–Flag fusion) and anti-HA antibody (which detects rhodopsin–HA fusion). The cross-linked products are marked with arrow heads, and free-arrestin and free-rhodopsin are indicated by asterisks. Arrestin (3A) and rhodopsin (4M) without cysteine mutations do not form cross-linked products. b, The cross-linked product of finger loop residue G77C with N3107.57C of TM7 was sensitive to treatment with reducing agents, indicating the cross-linking is mediated through disulfide bond formation. c, A close-up view of arrestin finger loop residues M76C and G77C and their cross-linking with rhodopsin, which shows that G77C was specifically cross-linked to N3107.57C of TM7 and Q3128.49 of helix 8, and M76C was cross-linked to N3107.57C of TM7 and Q3128.49C of helix 8, but not to other residues. d, Structure and cross-linking of finger loop N-terminal residues Q70C, E71C, and D72C of arrestin to T70C and K67C from ICL1 of rhodopsin. e, Structure and cross-linking of arrestin back loop residues R319C and T320C to Q237ICL3C from TM5 of rhodopsin.

Extended Data Figure 9 Structure comparison of the arrestin-bound rhodopsin with the β2-adrenergic receptor in complex with Gs protein (PDB code 3SN6) and the inactive rhodopsin (PDB code 1F88).

a, Superposition of arrestin-bound rhodopsin (green) with Gs protein-bound β2 adrenergic receptor (light yellow). The major conformational changes are indicated by arrows. b, An intracellular view of a superposition of arrestin-bound rhodopsin (green) and Gs protein-bound β2-adrenergic receptor (light yellow). c, Overlays of arrestin-bound rhodopsin (green) with inactive rhodopsin (pink) reveals specific conformational changes in each TM helix. The arrows indicate outward movements of TM helices. d, r.m.s.d. of Cα atom differences between arrestin-bound rhodopsin and inactive rhodopsin shows the large conformational changes in TM5 and TM6.

Extended Data Figure 10 Structure of rhodopsin-bound arrestin and its comparison with inactive and ‘pre-activated’ arrestin.

a, b, The charge potential surface map of rhodopsin from the rhodopsin–arrestin bound complex shows that the cytoplasmic rhodopsin TM bundle surface is positively charged (blue) whereas its C-terminal tail is negatively charged (red). c, d, Charged surface of arrestin from the rhodopsin–arrestin bound complex shows that the arrestin finger loop is negatively charged (red) and its N-terminal β-strand interface is positively charged (blue). The charge distribution in rhodopsin and arrestin is complementary to each other for their interactions. e, Comparison of rhodopsin-bound arrestin (light blue) with inactive arrestin (brown, PDB code: 1CF1), showing an ∼20° rotation between the N- and C- domains of arrestin. f, Comparison of rhodopsin-bound arrestin (dark brown) with pre-activated arrestin (light brown, PDB code 4J2Q), showing conformational changes in the finger loop, which adopts an α-helical conformation (cyan) in the complex. The extended finger loop conformation would protrude into the rhodopsin TM bundle and is not compatible with receptor binding. Computational model for the full rhodopsin–arrestin complex is shown in panels b and d.

Extended Data Figure 11 A computational model of phosphorylated rhodopsin in complex with arrestin and salt sensitivity of the rhodopsin–arrestin interaction.

a–d, An overall view (a) and close-up views (b–d) of the computational model of the rhodopsin C-tail with phospho-serine at positions 334, 338 and 343 in complex with arrestin. e, The AlphaScreen control (biotin–His6) shows much less salt sensitivity than the interaction between His-tag–rhodopsin and biotin arrestin, which is very sensitive to salt, with an IC50 of around 200 mM NaCl (100 mM NaCl added to 100 mM salt of the original assay buffer) (n = 3, error bars, s.d.).

Extended Data Figure 12 A positive charge property is commonly found at the cytoplasmic side of GPCRs.

a–e, Surface charge potential of the cytoplasmic side of selected agonist bound GPCR structures: β1AR, PDB code 2Y02 (a); β2AR, PDB code 3PDS (b); A2A adenosine receptor, PDB code 3QAK (c); serotonin receptor 5HT1B, PDB code 4IAR (d); serotonin receptor 5HT2B, PDB code 4IB4 (e). Positive and negative charge potentials are shown in blue and red, respectively. f, Sequence alignment of the finger loop region highlighting negatively charged residues (shown in red), which are conserved in all subtypes of arrestins.

Extended Data Figure 13 A possible role of the arrestin C-edge in lipid binding.

a, b, The asymmetric assembly of the rhodopsin–arrestin complex in the presence of a lipid membrane bilayer, showing the C-edge of arrestin dipping into the lipid layer. c, d, A close-up view of the C-edge of arrestin in the membrane layer, where the conserved hydrophobic side chains are shown. The figure was made using the computational model for the full rhodopsin–arrestin complex.

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Kang, Y., Zhou, X., Gao, X. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.Nature 523, 561–567 (2015). https://doi.org/10.1038/nature14656

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