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.

Cryo-EM structure of human rhodopsin bound to an inhibitory G protein

A Publisher Correction to this article was published on 21 June 2018

This article has been updated

Abstract

G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, Gi-bound and arrestin-bound forms of rhodopsin with inactive and Gs-bound forms of the β2-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of Gs, Gi and arrestin by activated G-protein-coupled receptors.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Assembly of the rhodopsin–Gi protein complex.
Fig. 2: The cryo-EM structure of the rhodopsin–Gi complex.
Fig. 3: The rhodopsin–Gi interface.
Fig. 4: Structural comparison of Gi-bound rhodopsin with inactive rhodopsin, arrestin-bound rhodopsin, and GαCT-bound rhodopsin.
Fig. 5: Structural comparison of Gi-bound rhodopsin with Gs-bound β2AR.
Fig. 6: TM6 dynamics of Gi- and Gs-coupled receptors.

Change history

  • 21 June 2018

    In the PDF version of this Article, owing to a typesetting error, an incorrect figure was used for Extended Data Fig. 5; the correct figure was used in the HTML version. This has been corrected online.

References

  1. 1.

    Fredriksson, R., Lagerström, M. C., Lundin, L. G. & Schiöth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003).

    Article  PubMed  CAS  Google Scholar 

  2. 2.

    Neves, S. R., Ram, P. T. & Iyengar, R. G protein pathways. Science 296, 1636–1639 (2002).

    ADS  Article  PubMed  CAS  Google Scholar 

  3. 3.

    Gurevich, E. V. & Gurevich, V. V. Arrestins: ubiquitous regulators of cellular signaling pathways. Genome Biol. 7, 236 (2006). 

    Article  PubMed  CAS  Google Scholar 

  4. 4.

    Zhou, X. E., Melcher, K. & Xu, H. E. Understanding the GPCR biased signaling through G protein and arrestin complex structures. Curr. Opin. Struct. Biol. 45, 150–159 (2017).

    Article  PubMed  CAS  Google Scholar 

  5. 5.

    Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

    ADS  Article  PubMed  CAS  Google Scholar 

  6. 6.

    Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. 7.

    Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273 (2007).

    ADS  Article  PubMed  CAS  Google Scholar 

  8. 8.

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

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. 9.

    Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474, 521–525 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. 10.

    Choe, H. W. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011).

    ADS  Article  PubMed  CAS  Google Scholar 

  11. 11.

    Wang, C. et al. Structural basis for molecular recognition at serotonin receptors. Science 340, 610–614 (2013).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 12.

    Standfuss, J. et al. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471, 656–660 (2011).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. 13.

    Rosenbaum, D. M. et al. Structure and function of an irreversible agonist–β2 adrenoceptor complex. Nature 469, 236–240 (2011).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. 14.

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

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. 15.

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

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. 16.

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

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. 17.

    Flock, T. et al. Selectivity determinants of GPCR–G-protein binding. Nature 545, 317–322 (2017).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. 18.

    Palczewski, K. G protein-coupled receptor rhodopsin. Annu. Rev. Biochem. 75, 743–767 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. 19.

    Zhou, X. E., Melcher, K. & Xu, H. E. Structure and activation of rhodopsin. Acta Pharmacol. Sin. 33, 291–299 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Hamm, H. E. How activated receptors couple to G proteins. Proc. Natl Acad. Sci. USA 98, 4819–4821 (2001).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. 21.

    Van Meurs, K. P. et al. Deduced amino acid sequence of bovine retinal G: similarities to other guanine nucleotide-binding proteins. Proc. Natl Acad. Sci. USA 84, 3107–3111 (1987).

    ADS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Lerea, C. L., Somers, D. E., Hurley, J. B., Klock, I. B. & Bunt-Milam, A. H. Identification of specific transducin α subunits in retinal rod and cone photoreceptors. Science 234, 77–80 (1986).

    ADS  Article  PubMed  CAS  Google Scholar 

  23. 23.

    Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523,561–567 (2015).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. 24.

    Zhou, X. E. et al. Identification of phosphorylation codes for arrestin recruitment by G protein-coupled receptors. Cell 170, 457–469 (2017).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. 25.

    Maeda, S. et al. Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein. PLoS One 9, e98714 (2014).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. 26.

    Liu, P. et al. The structural basis of the dominant negative phenotype of the Gαi1β1γ2 G203A/A326S heterotrimer. Acta Pharmacol. Sin. 37, 1259–1272 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. 27.

    Van Eps, N. et al. Conformational equilibria of light-activated rhodopsin in nanodiscs. Proc. Natl Acad. Sci. USA 114, E3268–E3275 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. 28.

    Van Eps, N. et al. Gi- and Gs-coupled GPCRs show different modes of G-protein binding. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1721896115 (2018).

  29. 29.

    Oldham, W. M., Van Eps, N., Preininger, A. M., Hubbell, W. L. & Hamm, H. E. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins. Nat. Struct. Mol. Biol. 13, 772–777 (2006).

    Article  PubMed  CAS  Google Scholar 

  30. 30.

    Oldham, W. M. & Hamm, H. E. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 9, 60–71 (2008).

    Article  PubMed  CAS  Google Scholar 

  31. 31.

    Marin, E. P., Krishna, A. G. & Sakmar, T. P. Rapid activation of transducin by mutations distant from the nucleotide-binding site: evidence for a mechanistic model of receptor-catalyzed nucleotide exchange by G proteins. J. Biol. Chem. 276, 27400–27405 (2001).

    Article  PubMed  CAS  Google Scholar 

  32. 32.

    Marin, E. P., Krishna, A. G. & Sakmar, T. P. Disruption of the α5 helix of transducin impairs rhodopsin-catalyzed nucleotide exchange. Biochemistry 41, 6988–6994 (2002).

    Article  PubMed  CAS  Google Scholar 

  33. 33.

    Garcia, P. D., Onrust, R., Bell, S. M., Sakmar, T. P. & Bourne, H. R. Transducin-α C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells. EMBO J. 14, 4460–4469 (1995).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  34. 34.

    Onrust, R. et al. Receptor and βγ binding sites in the alpha subunit of the retinal G protein transducin. Science 275, 381–384 (1997).

    Article  PubMed  CAS  Google Scholar 

  35. 35.

    Skiba, N. P., Bae, H. & Hamm, H. E. Mapping of effector binding sites of transducin alpha-subunit using Gαt/Gαi1 chimeras. J. Biol. Chem. 271, 413–424 (1996).

    Article  PubMed  CAS  Google Scholar 

  36. 36.

    Sun, D. et al. Probing Gαi1 protein activation at single-amino acid resolution. Nat. Struct. Mol. Biol. 22, 686–694 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. 37.

    Itoh, Y., Cai, K. & Khorana, H. G. Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent. Proc. Natl Acad. Sci. USA 98, 4883–4887 (2001).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. 38.

    Subramaniam, S., Gerstein, M., Oesterhelt, D. & Henderson, R. Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin. EMBO J. 12, 1–8 (1993).

    PubMed  PubMed Central  CAS  Article  Google Scholar 

  39. 39.

    Subramaniam, S. & Henderson, R. Molecular mechanism of vectorial proton translocation by bacteriorhodopsin. Nature 406, 653–657 (2000).

    ADS  Article  PubMed  CAS  Google Scholar 

  40. 40.

    Slessareva, J. E. et al. Closely related G-protein-coupled receptors use multiple and distinct domains on G-protein α-subunits for selective coupling. J. Biol. Chem. 278, 50530–50536 (2003).

    Article  PubMed  CAS  Google Scholar 

  41. 41.

    Kling, R. C., Lanig, H., Clark, T. & Gmeiner, P. Active-state models of ternary GPCR complexes: determinants of selective receptor–G-protein coupling. PloS One 8, (2013).

  42. 42.

    DeVree, B. T. et al. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs. Nature 535, 182–186 (2016).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. 43.

    Dror, R. O. et al. Signal transduction. Structural basis for nucleotide exchange in heterotrimeric G proteins. Science 348, 1361–1365 (2015).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. 44.

    Dickson, B. M., de Waal, P. W., Ramjan, Z. H., Xu, H. E. & Rothbart, S. B. A fast, open source implementation of adaptive biasing potentials uncovers a ligand design strategy for the chromatin regulator BRD4. J. Chem. Phys. 145, 154113 (2016).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. 45.

    Huang, W. J. et al. Structural insights into μ-opioid receptor activation. Nature 524, 315–321 (2015).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. 46.

    Rose, A. S. et al. Position of transmembrane helix 6 determines receptor G protein coupling specificity. J. Am. Chem. Soc. 136, 11244–11247 (2014).

  47. 47.

    Caro, L. N. et al. Rapid and facile recombinant expression of bovine rhodopsin in HEK293S GnTI cells using a PiggyBac inducible system. Methods Enzymol. 556, 307–330 (2015).

    Article  PubMed  CAS  Google Scholar 

  48. 48.

    Hornsby, M. et al. A high through-put platform for recombinant antibodies to folded proteins. Mol. Cell Proteomics 14, 2833–2847 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. 49.

    Paduch, M. & Kossiakoff, A. A. Generating conformation and complex-specific synthetic antibodies. Methods Mol. Biol. 1575, 93–119 (2017).

    Article  PubMed  CAS  Google Scholar 

  50. 50.

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. 51.

    Brilot, A. F. et al. Beam-induced motion of vitrified specimen on holey carbon film. J. Struct. Biol. 177, 630–637 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. 52.

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  Google Scholar 

  53. 53.

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  PubMed  CAS  Google Scholar 

  54. 54.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  CAS  Google Scholar 

  55. 55.

    Wang, R. Y. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, e17219 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. 57.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    Article  PubMed  CAS  Google Scholar 

  58. 58.

    Manglik, A. et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161, 1101–1111 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. 59.

    Šali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  PubMed  Google Scholar 

  60. 60.

    Mahalingam, M., Martínez-Mayorga, K., Brown, M. F. & Vogel, R. Two protonation switches control rhodopsin activation in membranes. Proc. Natl Acad. Sci. USA 105, 17795–17800 (2008).

    ADS  Article  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Ranganathan, A., Dror, R. O. & Carlsson, J. Insights into the role of Asp792.50 in β2 adrenergic receptor activation from molecular dynamics simulations. Biochemistry 53, 7283–7296 (2014).

    Article  PubMed  CAS  Google Scholar 

  62. 62.

    Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).

    Article  PubMed  CAS  Google Scholar 

  63. 63.

    Zoete, V., Cuendet, M. A., Grosdidier, A. & Michielin, O. SwissParam: a fast force field generation tool for small organic molecules. J. Comput. Chem. 32, 2359–2368 (2011).

    Article  PubMed  CAS  Google Scholar 

  64. 64.

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

    ADS  Article  PubMed  CAS  Google Scholar 

  65. 65.

    Dickson, B. M., de Waal, P. W., Ramjan, Z. H., Xu, H. E. & Rothbart, S. B. A fast, open source implementation of adaptive biasing potentials uncovers a ligand design strategy for the chromatin regulator BRD4. J. Chem. Phys. 145, 154113 (2016).

    ADS  Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. 66.

    Dickson, B. M. Overfill protection and hyperdynamics in adaptively biased simulations. J. Chem. Theory Comput. 13, 5925–5932 (2017).

    Article  PubMed  CAS  Google Scholar 

  67. 67.

    Kohlhoff, K. J. et al. Cloud-based simulations on Google Exacycle reveal ligand modulation of GPCR activation pathways. Nat. Chem. 6, 15–21 (2014).

    Article  PubMed  CAS  Google Scholar 

  68. 68.

    Alexander, S. P. H. et al. The Concise Guide To Pharmacology 2017/18: G protein-coupled receptors. Br. J. Pharmacol. 174 (Suppl. 1), S17–S129 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

Cryo-EM data were collected at the David Van Andel Advanced Cryo-Electron Microscopy Suite in the Van Andel Research Institute. This work was supported in part by the National Institutes of Health grant, DK071662, American Asthma Foundation, Jay and Betty Van Andel Foundation, Ministry of Science and Technology (China) grants 2012ZX09301001 and 2012CB910403, 2013CB910600, XDB08020303, 2013ZX09507001 (to H.E.X.), GM117372 (to A.K.), GM0875119 (to A.A.K.), grant from Pfizer (to A.A.K.), the National Natural Science Foundation 31770796 (to Y.J.), the Canada Excellence Research Chairs program (to O.P.E.), the Canadian Institute for Advanced Research (to O.P.E.), the Anne and Max Tanenbaum Chair in Neuroscience (to O.P.E.), by funds from the Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD (to S.S.), and by federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. We thank H. Li and W. Lü for help with analysing the cryo-EM data and for advice on refinement, L. Bai and Z. Yuan for advice on 3D reconstruction, V. Falconieri for assistance with figure preparation, the HPC team at VARI for computational support, D. Nadziejka for manuscript editing, and B. Dickson for consultation on molecular dynamics simulation.

Author information

Affiliations

Authors

Contributions

Y.K. initiated the project, prepared samples, performed data acquisition and structure determination, and prepared the figures and manuscript writing; H.E.X. and K.M. conceived the project and designed the research, and wrote the paper with contributions from all authors; O.K., X.E.Z., A.B. and S.S. performed image processing, structure determination, figure preparation, and manuscript writing; P.W.d.W. performed computational experiments, analysed the structure, prepared figures, and manuscript writing; P.D., S.M., S.E. and A.A.K. designed and performed Fab selection; N.V.E., T.M. and O.P.E. designed and performed DEER experiments; X.G., Y.Y., P.L. and Y.J. performed cell-based assays; G.Z. and X.M. helped with data collection.

Corresponding authors

Correspondence to Anthony A. Kossiakoff, Sriram Subramaniam or H. Eric Xu.

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.

Extended data figures and tables

Extended Data Fig. 1 Purification, characterization and cryo-EM images of the Rho–Gi–Fab complex.

a, Representative elution profile of the purified Rho–Gi–Fab_G50 complex on Superdex 200 10/300 gel filtration. b, SDS–PAGE analysis of the complex after gel filtration. c, The inability of rhodopsin to stimulate the Gs-mediated signalling as assayed by the cAMP-driven luciferase reporter assays. The glucagon-like peptide 1 receptor (GLP-1R) shows stronger Gs-meditated signalling with the agonist GLP-1 (n = 3 independent experiments). Data are mean ± s.d. d, An overall view of rhodopsin showing the three intramolecular distances between two nitroxide N–O bonds based on the models of the R1 nitroxide pairs Y74R1-Q225R1, Y74R1-R252R1 and Y74R1-M308R1, respectively (Y742.41, Q2255.60, R2526.35, M3087.55; superscripts denote Ballesteros–Weinstein numbering). R1 side-chain modelling details have been described previously27. e, Similar DEER distance distributions of TM6 and TM7 to TM2 of rhodopsin bound to Gi and Gt. f, Time domain data of DEER measurements.

Extended Data Fig. 2 Cryo-EM images and single-particle analysis  of the Rho–Gi–Fab complex.

a, Representative cryo-EM micrograph of Rho–Gi–Fab complex. Examples of particle projections are circled. b, Reference-free two-dimensional class averages of the complex in digitonin micelles. c, Half-map Fourier shell correlation (FSC) plots as produced by RELION with the mask used shown as an inset. d, FSC curve of model versus the full map, as well as FSC curves obtained for a model refined against a half-map and compared to the two half-maps as well as the full model. The r.m.s.d. between the model refined against half-map and compared to the full map, and the one refined against the full map is 0.984 Å, and their corresponding FSCs against the final map show a resolution difference at the 0.5-cutoff of approximately 0.1 Å. e, Particle classification and refinement. f, Local resolution map of the rhodopsin–Gi complex.

Extended Data Fig. 3 Electron microscopy density map of rhodopsin–Gi complex.

ac, Three views of the electron microscopy density map of the rhodopsin–Gαi interface. d, Electron microscopy density map of all rhodopsin transmembrane helices and helix 8. eg, An overall view of the rhodoposin–Gαi interface (e), and electron microscopy density map for the TM6 of rhodopsin (f) and the α5-helix of Gαi (g).

Extended Data Fig. 4 The rhodopsin–Gi interface and disulfide crosslinking of rhodopsin with Gαi.

a, The rhodopsin–Gi interface surrounding the G352 residue of Gαi α5-helix. Not all side chains shown are visible in the map but shown here for illustrating their Cα positions to facilitate understanding of data in panel b. b, Lack of disulfide crosslinking of G352C of Gi with surrounding residues from rhodopsin (compare with dn = 3 independent experiments). c, Interactions at the interface between ICL2 of rhodopsin and αN helix of Gαi. The side chains are not visible in the map but shown here for illustrating their Cα positions. d, Demonstration that E28C of Gαi can be disulfide cross-linked to rhodopsin residues N145CICL2 and F146C ICL2 (n = 3 independent experiments).

Extended Data Fig. 5 Structural comparison of Gi-bound rhodopsin, Gs-bound  GLP-1R, and Gs-bound  CTR, and the role of α4-helix of Gα in receptor selectivity.

a, b, Side and cytoplasmic views of Gi-bound rhodopsin (orange) overlaid with Gs-bound GLP-1R (PDB code 5VAI, light blue, black arrows indicate differences in helix positions). c, d, Side and cytoplasmic views of Gi-bound rhodopsin (orange) overlaid with Gs-bound CTR (PDB code 5UZ7, grey). e, f, Side-by-side comparison of the rhodopsin–Gi complex (e) with the β2AR–Gs complex (f). g. An overlay of the rhodopsin–Gi complex with the β2AR–Gs complex reveals possible collision of TM5 of β2AR with α4-helix of Gαi.

Extended Data Fig. 6 The mechanism of rhodopsin-mediated Gi activation.

a, b, Superposition of the rhodopsin–Gi complex with the inactive GDP-bound Gi (PDB code 1GG2) reveals separation of the AHD from the Ras domain of Gαi (a) and conformational changes in the α5-helix (b). c, d, Side-by-side comparison of the GDP-binding site of the Gαi Ras domain in the inactive GDP-bound Gαi (c) and nucleotide-free state Gαi with GDP added for comparison (d).

Extended Data Fig. 7 Collective variables for mABP simulations and free-energy landscapes of mABP simulations.

a, To bias movement between TM6 relative to that of the receptor bundle, two centre-of-geometry (COG) distance collective variables (CVs) were implemented into fABMACS66. CV1 and CV2 are COG distances between selected atoms of TM6 to TM1/2 and TM6 to TM3/4 respectively. Collective variable atoms for the rhodopsin simulation are highlighted. b, COG collective variable formula and the CV1 and CV2 distances. c, Potential energy surface reveals that CV1 and CV2 distances are larger in the Gs-coupled receptors (A2AR and β2AR) than those in the Gi-coupled receptors (mOR1 and rhodopsin).

Extended Data Fig. 8 Enrichment profiles for Gi and Gs coupling receptors.

ac, Relative probability of hydrophobic and polar residues for Gi (n = 76) and Gs (n = 25) coupling receptors. Residues with relative enrichments over 20% were mapped onto the structures of Gs-bound β2AR (b) and Gi-bound rhodopsin (c). GPCR principal coupling was previously defined68. df, Interaction network of TM6.36 of β2AR, A2AR and rhodopsin with the G protein α5-helix. g, Hydrogen bonding between TM3.36 and the backbone of TM6.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Extended Data Table 2 GPCR simulation systems used in the current study

Supplementary information

Reporting Summary

Video 1: Structural difference between inactive Rho and Gi-bound Rho.

Conformational changes in the transmembrane helices of rhodopsin illustrated by morphing from inactive state to Gi-bound state.

Video 2: Structural difference between arrestin-bound Rho and Gi-bound Rho.

Conformational changes in the transmembrane helices of rhodopsin illustrated by morphing from arrestin-bound state to Gi-bound state.

Video 3: Structural difference between Gs-bound β2-AR and Gi-bound Rho.

Conformational changes in the transmembrane helices of GPCR illustrated by morphing from β2-AR in Gs-bound state to rhodopsin in Gi-bound state.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kang, Y., Kuybeda, O., de Waal, P.W. et al. Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature 558, 553–558 (2018). https://doi.org/10.1038/s41586-018-0215-y

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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