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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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.
Extended data figures and tables
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.
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.
a–c, 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. e–g, 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).
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 d; n = 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.
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).
a–c, 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. d–f, 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.
Conformational changes in the transmembrane helices of rhodopsin illustrated by morphing from inactive state to Gi-bound state.
Conformational changes in the transmembrane helices of rhodopsin illustrated by morphing from arrestin-bound state to Gi-bound state.
Conformational changes in the transmembrane helices of GPCR illustrated by morphing from β2-AR in Gs-bound state to rhodopsin in Gi-bound state.