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Structural basis and mechanism of activation of two different families of G proteins by the same GPCR

Nature Structural & Molecular Biology volume 28, pages 936–944 (2021)Cite this article

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Abstract

The β1-adrenergic receptor (β1-AR) can activate two families of G proteins. When coupled to Gs, β1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey β1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with β1-AR, the overall interacting modes between β1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by β1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).

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Fig. 1: Cryo-EM structure of the complex of isoproterenol-bound β1-AR and Gi.
Fig. 2: Structural basis of the activation of Gi by β1-AR.
Fig. 3: Functional studies of the interacting residues in β1-AR for the activation of Gi.
Fig. 4: Comparative structural analysis of the complexes of isoproterenol–β1-AR–Gi and isoproterenol–β1-AR–Gs.
Fig. 5: Comparison of the detailed interactions between β1-AR and Gαi and between β1-AR and Gαs.
Fig. 6: Cryo-EM structure of the complex of isoproterenol-bound β1-AR and Gi/s.
Fig. 7: Functional studies of the receptor regions contributing to the G-protein activating efficacy.

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Data availability

The cryo-EM reconstructions of the β1-AR–Gi–isoproterenol complex and the β1-AR–Gi/s–isoproterenol complex have been deposited in the Election Microscopy Data Bank (EMDB) under ID codes EMD-24789 and EMD-24790, respectively. The corresponding atomic models have been deposited in the Protein Data Bank (PDB) under ID codes 7S0F and 7S0G, respectively. Source data are provided with this paper.

References

  1. Benovic, J. L. Novel β2-adrenergic receptor signaling pathways. J. Allergy Clin. Immunol. 110, S229–S235 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Post, S. R., Hammond, H. K. & Insel, P. A. Beta-adrenergic receptors and receptor signaling in heart failure. Annu. Rev. Pharmacol. Toxicol. 39, 343–360 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Lohse, M. J., Engelhardt, S. & Eschenhagen, T. What is the role of β-adrenergic signaling in heart failure? Circ. Res. 93, 896–906 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Hilger, D., Masureel, M. & Kobilka, B. K. Structure and dynamics of GPCR signaling complexes. Nat. Struct. Mol. Biol. 25, 4–12 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Asano, T., Katada, T., Gilman, A. G. & Ross, E. M. Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by β-adrenergic receptors in reconstituted phospholipid vesicles. J. Biol. Chem. 259, 9351–9354 (1984).

    Article  CAS  PubMed  Google Scholar 

  6. Kompa, A. R., Gu, X. H., Evans, B. A. & Summers, R. J. Desensitization of cardiac β-adrenoceptor signaling with heart failure produced by myocardial infarction in the rat. Evidence for the role of Gi but not Gs or phosphorylating proteins. J. Mol. Cell. Cardiol. 31, 1185–1201 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Wang, J. et al. Gαi is required for carvedilol-induced β1 adrenergic receptor β-arrestin biased signaling. Nat. Commun. 8, 1706 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Dwivedi, H., Baidya, M. & Shukla, A. K. GPCR signaling: the interplay of Gαi and β-arrestin. Curr. Biol. 28, R324–R327 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Lukasheva, V. et al. Signal profiling of the β1AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β1AR and β2AR. Sci. Rep. 10, 8779 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rubenstein, R. C., Linder, M. E. & Ross, E. M. Selectivity of the β-adrenergic receptor among Gs, Gi’s, and Go: assay using recombinant α subunits in reconstituted phospholipid vesicles. Biochemistry 30, 10769–10777 (1991).

    Article  CAS  PubMed  Google Scholar 

  11. Su, M. et al. Structural basis of the activation of heterotrimeric Gs-protein by isoproterenol-bound β1-adrenergic receptor. Mol. Cell 80, 59–71 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang, J., Chen, S., Zhang, J. J. & Huang, X. Y. Crystal structure of oligomeric β1-adrenergic G protein-coupled receptors in ligand-free basal state. Nat. Struct. Mol. Biol. 20, 419–425 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wall, M. A. et al. The structure of the G protein heterotrimer Giα1β1γ2. Cell 83, 1047–1058 (1995).

    Article  CAS  PubMed  Google Scholar 

  14. Sprang, S. R., Chen, Z. & Du, X. Structural basis of effector regulation and signal termination in heterotrimeric Gα proteins. Adv. Protein Chem. 74, 1–65 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Noel, J. P., Hamm, H. E. & Sigler, P. B. The 2.2-Å crystal structure of transducin-α complexed with GTPγS. Nature 366, 654–663 (1993).

    Article  CAS  PubMed  Google Scholar 

  16. Coleman, D. E. et al. Structures of active conformations of Giα1 and the mechanism of GTP hydrolysis. Science 265, 1405–1412 (1994).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kang, Y. et al. Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature 558, 553–558 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wall, M. A., Posner, B. A. & Sprang, S. R. Structural basis of activity and subunit recognition in G protein heterotrimers. Structure 6, 1169–1183 (1998).

    Article  CAS  PubMed  Google Scholar 

  20. Aurora, R., Srinivasan, R. & Rose, G. D. Rules for α-helix termination by glycine. Science 264, 1126–1130 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Liu, X. et al. Structural insights into the process of GPCR-G protein complex formation. Cell 177, 1243–1251 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sprang, S. R. G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639–678 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Lee, E., Taussig, R. & Gilman, A. G. The G226A mutant of Gsα highlights the requirement for dissociation of G protein subunits. J. Biol. Chem. 267, 1212–1218 (1992).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Koehl, A. et al. Structure of the μ-opioid receptor–Gi protein complex. Nature 558, 547–552 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Krishna Kumar, K. et al. Structure of a signaling cannabinoid receptor 1-G protein complex. Cell 176, 448–458 e412 (2019).

    Article  CAS  PubMed  Google Scholar 

  28. Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D. & Bourne, H. R. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature 363, 274–276 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Conklin, B. R. & Bourne, H. R. Structural elements of Gα subunits that interact with Gβγ, receptors, and effectors. Cell 73, 631–641 (1993).

    Article  CAS  PubMed  Google Scholar 

  30. Hisano, Y. et al. Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes. J. Exp. Med. 216, 1582–1598 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jelinek, V., Mosslein, N. & Bunemann, M. Structures in G proteins important for subtype selective receptor binding and subsequent activation. Commun. Biol. 4, 635 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Okashah, N. et al. Variable G protein determinants of GPCR coupling selectivity. Proc. Natl Acad. Sci. USA 116, 12054–12059 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Draper-Joyce, C. J. et al. Structure of the adenosine-bound human adenosine A1 receptor–Gi complex. Nature 558, 559–563 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Ma, X. et al. Analysis of β2AR-Gs and β2AR-Gi complex formation by NMR spectroscopy. Proc. Natl Acad. Sci. USA 117, 23096–23105 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bos, J. L., Rehmann, H. & Wittinghofer, A. GEFs and GAPs: critical elements in the control of small G proteins. Cell 129, 865–877 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D. & Kuriyan, J. The structural basis of the activation of Ras by Sos. Nature 394, 337–343 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Bourne, H. R., Sanders, D. A. & McCormick, F. The GTPase superfamily: conserved structure and molecular mechanism. Nature 349, 117–127 (1991).

    Article  CAS  PubMed  Google Scholar 

  38. Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Baker, J. G. A full pharmacological analysis of the three turkey β-adrenoceptors and comparison with the human β-adrenoceptors. PLoS ONE 5, e15487 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Baker, J. G., Proudman, R. G. & Tate, C. G. The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (β36) deletions essential for crystallisation of the turkey β-adrenoceptor. Naunyn Schmiedebergs Arch. Pharm. 384, 71–91 (2011).

    Article  CAS  Google Scholar 

  41. Carragher, B. et al. Leginon: an automated system for acquisition of images from vitreous ice specimens. J. Struct. Biol. 132, 33–45 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  43. Scheres, S. H. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016).

    Article  CAS  PubMed  Google Scholar 

  44. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

  46. Terwilliger, T. C., Ludtke, S. J., Read, R. J., Adams, P. D. & Afonine, P. V. Improvement of cryo-EM maps by density modification. Nat. Methods 17, 923–927 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  51. McEwen, D. P., Gee, K. R., Kang, H. C. & Neubig, R. R. Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins. Anal. Biochem. 291, 109–117 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Wan, Y., Kurosaki, T. & Huang, X. Y. Tyrosine kinases in activation of the MAP kinase cascade by G-protein-coupled receptors. Nature 380, 541–544 (1996).

    Article  CAS  PubMed  Google Scholar 

  53. Ma, Y. C. & Huang, X. Y. Identification of the binding site for Gqα on its effector Bruton’s tyrosine kinase. Proc. Natl Acad. Sci. USA 95, 12197–12201 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of our research groups for helpful discussions and comments on the manuscript. We thank A. Inoue (Tohoku University, Japan) for the Gα-depleted HEK293 cells. This work was supported by NIH grant no. GM138676 (X.-Y.H.), the Josie Robertson Investigators Program (R.K.H.) and the Searle Scholars Program (R.K.H.). The Simons Electron Microscopy Center and the National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center are supported by grants from the NIH National Institute of General Medical Sciences (GM103310), NYSTAR and the Simons Foundation (SF349247). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Kamela O. Alegre, Navid Paknejad, Minfei Su.

Authors and Affiliations

  1. Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY, USA

    Kamela O. Alegre, Minfei Su, Jian-Shu Lou, Jianyun Huang, Joel R. Meyerson & Xin-Yun Huang

  2. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Navid Paknejad & Richard K. Hite

  3. Simons Electron Microscopy Center, National Resource for Automated Molecular Microscopy, New York Structural Biology Center, New York, NY, USA

    Kelsey D. Jordan & Edward T. Eng

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Contributions

K.O.A. expressed and purified β1-AR, Gαi, Gβ1γ2 and the protein complexes, made cryo-EM grids, performed cryo-EM screening, data collection, image processing, determined the EM density map and performed the functional studies. N.P. made cryo-EM grids, performed data collection, image processing, EM density map determination and model building. M.S. purified β1-AR and the complex of β1-AR–Gi, provided the β1-AR–Gs structural information and prepared some figures. J.-S.L. performed the functional studies. J.H. generated the β1-AR and G-protein constructs. K.D.J. and E.T.E. performed negative-stain EM and data collection. J.R.M. performed image processing. R.K.H. supervised the project and performed image processing, EM density map determination, model building and data interpretation. X.-Y.H. supervised the project, interpreted data and wrote the manuscript. All authors contributed to the final version of the manuscript.

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Correspondence to Richard K. Hite or Xin-Yun Huang.

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The authors declare no competing interests.

Additional information

Peer review information Nature Structural & Molecular Biology thanks Qing Fan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Summary of cryo-EM data processing.

Details are found in the methods. In short, 2680 micrographs were collected on a Krios equipped with Gatan K2 direct electron detector. Heterogeneous refinement was used to remove junk, with 2D classification as a form of verification. Once an ideal particle stack was identified, subsequent rounds of heterogeneous refinement were combined with local CTF refinement and Bayesian polishing to further improve the map. Local refinements of b1-AR and Gi helped bring out features in the periphery of the structure. The model was built into the consensus and local refinements, and then used to generate a composite map that was used for the final round of real-space refinement. Overall, even in the consensus structure, almost all of the model was well represented by density. The alpha-helical domain was less well resolved due to flexibility, but intermediate resolution rigid-body docking was possible.

Extended Data Fig. 2 Per residue model-map correlation coefficient plots.

Per residue model-map correlation coefficient was calculated using phenix.real_space_refine with maps pre (red line) and post (black line) density modification. In both β1-AR–Gi complex and β1-AR–Gi/s complex models, chain A is the Ras-like domain of Gα, Chain B is Gβ1, Chain G is Gγ2, Chain HD is the helical domain of Gα, and Chain R is β1-AR. Comparing model-map correction coefficient pre and post density modification, especially for β1-AR–Gi complex, density modification shows a clear improvement across all chains.

Extended Data Fig. 3 Comparison of the inactive β1-AR receptor with the activated β1-AR in complex with Gi.

(a-c) Side (left), extracellular (top right) and cytoplasmic (bottom right) views of the inactive β1-AR (grey)(PDB code 4GPO) compared to the activated β1-AR in complex with Gi. (d) Structural comparison of the Gi α5-helix binding region to show the major conformational changes of β1-AR. (e) The ionic lock between Arg139 and Glu285 is broken in the active state. (f) Tyr343 packs against Leu132 and Ile135 in the active state. (g) Cryo-EM density map of the Gi α5-helix binding region (contoured at 1.2 σ level).

Extended Data Fig. 4 Isoproterenol occupancy in orthosteric binding pocket of β1-AR.

(a,b) Side view (a) and oblique view (b) of activated β1-AR to highlight the location of the agonist isoproterenol bound in the orthosteric binding pocket at the extracellular side of the receptor. β1-AR is shown in cartoon in red and isoproterenol is shown in green. Experimental density carved around ligand shown at 13σ, supporting complete occupancy. (c) Close-up view of the orthosteric binding pocket with model docked into 8σ experimental density to highlight agonist coordination. Isoproterenol is colored in green and residues lining the binding pocket are in red.

Extended Data Fig. 5 Rotational opening of the α-helical domain during G-protein activation by GPCRs.

(a) Structure of Gαi in the complex of β1-AR–Gi shows the rotational opening of the α-helical domain away from the Ras-like domain. (b) Comparison of the structures of Gαi in the complex of β1-AR–Gi (in green and orange) and in the inactive GDP-bound Gi crystal structure (in gray; PDB: 1GG2). (c) View from the receptor towards the cytoplasmic end shows the rotation of the α-helical domain from the position in inactive Gi (in gray) to the location in the β1-AR–Gi complex (in orange). (d) View from Gβγ towards the Ras-like domain shows the position of the α-helical domain relative to Gβ. (e and f) Comparisons of the locations of the α-helical domains in the complexes of β2-AR–Gs (PDB: 3SN6), β1-AR–Gs (PDB: 7JJO), rhodopsin–Gi (PDB: 6CMO), and β1-AR–Gi (this paper).

Extended Data Fig. 6 Conformational changes of the GDP/GTP-binding pocket of Gαi after β1-AR interaction.

(a) Comparison of the β1 strand, α1-helix and the β1-α1 loop of the Ras-like domains from β1-AR–Gi (in green) and from Gαi1Gβ1Gγ2 (PDB: 1GG2; in gray) when the Ras-like domains are superimposed. (b) Comparison of Switch II region from β1-AR–Gi and from Gαi1Gβ1Gγ2. (c) Comparison of Switch III region from β1-AR–Gi and from Gαi1Gβ1Gγ2. (d) Comparison of the regions from αG to α5-helix from β1-AR–Gi and from Gαi1Gβ1Gγ2. (E) Comparison of all GDP-interacting residues of the Ras-like domains from β1-AR–Gi and from Gαi1Gβ1Gγ2.

Extended Data Fig. 7 Structural changes of G-proteins during the activation.

Separation of the Ras-like domains of Gi (a) and Gs (b) from Gβγ subunits in the complex of β1-AR–Gi (a) and β1-AR–Gs (b). Gβγ subunits are superimposed.

Extended Data Fig. 8 Conformational changes of the GDP-binding pocket of Gαs after β1-AR interaction.

(a) Comparison of the β1 strand, α1-helix and the β1-α1 loop of the Ras-like domains from β1-AR–Gs (in violet) and from GαsGβ1Gγ2 (PDB: 6EG8; in teal) when the Ras-like domains are superimposed. (b) Comparison of Switch II region from β1-AR–Gs and from GαsGβ1Gγ2. (c) Comparison of Switch III region from β1-AR–Gs and from GαsGβ1Gγ2. (d) Comparison of the regions from αG to α5-helix from β1-AR–Gs and from GαsGβ1Gγ2. (e) Comparison of all GDP-interacting residues of the Ras-like domains from β1-AR–Gs and from GαsGβ1Gγ2.(f) Disruptions of intra-molecular interactions of Gαs during Gs activation by β1-AR. An interaction between the sidechain of His373 in the α5-helix and the backbone of Arg38 in the αN-helix is broken. An interacting network involving the sidechain of Gln59 in the α1-helix, the backbone carbonyl of Ala352 in the β6-α5 loop, and the sidechain of Thr355 in the α5-helix is disrupted.

Extended Data Fig. 9 Summary of cryo-EM data processing.

Details are found in the methods. In short, 3,285 micrographs were collected on a Krios equipped with Gatan K2 direct electron detector. Heterogeneous refinement was used to remove junk, with 2D classification as a form of verification. Once an ideal particle stack was identified, subsequent rounds of heterogeneous refinement were combined with local CTF refinement and Bayesian polishing to further improve the map. Local refinement of Gi/s helped bring out features in the periphery of the structure. The model was built into the consensus and local refinements, and then used to generate a composite map that was used for the final round of real-space refinement.

Extended Data Fig. 10 Structural comparisons of the β1-AR–Gi complex with other GPCR–Gi complexes.

(a) The structure of β1-AR–Gi1 (in green) is superimposed with rhodopsin–Gi1 (PDB 6CMO, in magenta), cannabinoid receptor 1–Gi1 (PDB 6N4B, in cyan), and mu-opioid receptor–Gi1 (PDB 6DDE, in yellow). (b) Comparisons of the Gαi1Gβγ trimer structures in the four complexes (superimposed by Gβ subunits). (c) Comparisons of the interactions between ICL2s of the receptors and the αN-helices of Gαi1. (d) Comparisons of the interactions between the α5-helices of Gαi1 and the receptors. (e) Structural comparison of the regions around G203 of Gαi1 in the four structures.

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Alegre, K.O., Paknejad, N., Su, M. et al. Structural basis and mechanism of activation of two different families of G proteins by the same GPCR. Nat Struct Mol Biol 28, 936–944 (2021). https://doi.org/10.1038/s41594-021-00679-2

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