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.

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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.


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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

Author notes

  1. These authors contributed equally: Yanyong Kang, Oleg Kuybeda, Parker W. de Waal.


  1. Center for Cancer and Cell Biology, Innovation and Integration Program, Van Andel Research Institute, Grand Rapids, MI, USA

    • Yanyong Kang
    • , Parker W. de Waal
    • , X. Edward Zhou
    • , Xin Gu
    • , Yanting Yin
    • , Karsten Melcher
    •  & H. Eric Xu
  2. Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA

    • Oleg Kuybeda
    • , Alberto Bartesaghi
    •  & Sriram Subramaniam
  3. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA

    • Somnath Mukherjee
    • , Przemyslaw Dutka
    • , Satchal Erramilli
    •  & Anthony A. Kossiakoff
  4. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    • Ned Van Eps
    • , Takefumi Morizumi
    •  & Oliver P. Ernst
  5. Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

    • Przemyslaw Dutka
  6. University of Chinese Academy of Sciences, Beijing, China

    • Ping Liu
  7. Key Laboratory of Receptor Research, VARI-SIMM Center, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    • Ping Liu
    • , Yi Jiang
    •  & H. Eric Xu
  8. David Van Andel Advanced Cryo-Electron Microscopy Suite, Van Andel Research Institute, Grand Rapids, MI, USA

    • Xing Meng
    •  & Gongpu Zhao
  9. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    • Oliver P. Ernst
  10. Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA

    • Anthony A. Kossiakoff
  11. Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA

    • Sriram Subramaniam


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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.

Competing interests

The authors declare no competing interests.

Corresponding authors

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

Extended data figures and tables

  1. 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.

  2. 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.

  3. 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).

  4. 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).

  5. 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.

  6. 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).

  7. 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).

  8. 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.

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

Supplementary information

  1. Reporting Summary

  2. 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.

  3. 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.

  4. 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.

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