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Phase-plate cryo-EM structure of a class B GPCR–G-protein complex

Abstract

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαsβγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.

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Figure 1: The sCT–CTR–Gs cryo-EM structure.
Figure 2: The CTR transmembrane bundle orthosteric peptide-binding site.
Figure 3: Comparisons of inactive class B and the activated CTR structures.
Figure 4: CTR–G-protein interactions.
Figure 5: Comparison of a class A and class B GPCR Gs ternary complex.

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References

  1. Congreve, M. & Marshall, F. The impact of GPCR structures on pharmacology and structure-based drug design. Br. J. Pharmacol . 159, 986–996 (2010)

    CAS  PubMed  Article  Google Scholar 

  2. Kenakin, T. & Miller, L. J. Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery. Pharmacol. Rev. 62, 265–304 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Zhang, D., Zhao, Q. & Wu, B. Structural studies of G protein-coupled receptors. Mol. Cells 38, 836–842 (2015)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  5. Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013)

    CAS  Article  ADS  PubMed  Google Scholar 

  6. Jazayeri, A. et al. Extra-helical binding site of a glucagon receptor antagonist. Nature 533, 274–277 (2016)

    CAS  Article  ADS  PubMed  Google Scholar 

  7. Siu, F. Y. et al. Structure of the human glucagon class B G-protein-coupled receptor. Nature 499, 444–449 (2013)

    CAS  PubMed  Article  ADS  Google Scholar 

  8. Culhane, K. J., Liu, Y., Cai, Y. & Yan, E. C. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors. Front. Pharmacol. 6, 264 (2015)

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  9. Pal, K., Melcher, K. & Xu, H. E. Structure and mechanism for recognition of peptide hormones by Class B G-protein-coupled receptors. Acta Pharmacol. Sin. 33, 300–311 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Poyner, D. R. et al. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors. Pharmacol. Rev . 54, 233–246 (2002)

    CAS  PubMed  Article  Google Scholar 

  11. Bai, X. C., McMullan, G. & Scheres, S. H. How cryo-EM is revolutionizing structural biology. Trends Biochem. Sci. 40, 49–57 (2015)

    CAS  PubMed  Article  Google Scholar 

  12. De Zorzi, R., Mi, W., Liao, M. & Walz, T. Single-particle electron microscopy in the study of membrane protein structure. Microscopy 65, 81–96 (2016)

    CAS  PubMed  Article  Google Scholar 

  13. Danev, R., Tegunov, D. & Baumeister, W. Using the Volta phase plate with defocus for cryo-EM single particle analysis. eLife 6, e23006 (2017)

    PubMed  PubMed Central  Article  Google Scholar 

  14. Khoshouei, M., Radjainia, M., Baumeister, W. & Danev, R. Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate. Preprint at https://doi.org/10.1101/087841 (2016)

  15. Khoshouei, M. et al. Volta phase plate cryo-EM of the small protein complex Prx3. Nat. Commun. 7, 10534 (2016)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  16. Hilton, J. M., Dowton, M., Houssami, S. & Sexton, P. M. Identification of key components in the irreversibility of salmon calcitonin binding to calcitonin receptors. J. Endocrinol. 166, 213–226 (2000)

    CAS  PubMed  Article  Google Scholar 

  17. Furness, S. G. et al. Ligand-dependent modulation of G protein conformation alters drug efficacy. Cell 167, 739–749.e11 (2016)

    Article  CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Westfield, G. H. et al. Structural flexibility of the G α s α-helical domain in the β2-adrenoceptor Gs complex. Proc. Natl Acad. Sci. USA 108, 16086–16091 (2011)

    CAS  PubMed  Article  ADS  PubMed Central  Google Scholar 

  20. Van Eps, N. et al. Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit. Proc. Natl Acad. Sci. USA 108, 9420–9424 (2011)

    CAS  PubMed  Article  ADS  PubMed Central  Google Scholar 

  21. Andreotti, G. et al. Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic α-helix. J. Biol. Chem. 281, 24193–24203 (2006)

    CAS  PubMed  Article  Google Scholar 

  22. Johansson, E. et al. Type II turn of receptor-bound salmon calcitonin revealed by X-ray crystallography. J. Biol. Chem. 291, 13689–13698 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Ho, H. H., Gilbert, M. T., Nussenzveig, D. R. & Gershengorn, M. C. Glycosylation is important for binding to human calcitonin receptors. Biochemistry 38, 1866–1872 (1999)

    CAS  PubMed  Article  Google Scholar 

  24. Dods, R. L. & Donnelly, D. The peptide agonist-binding site of the glucagon-like peptide-1 (GLP-1) receptor based on site-directed mutagenesis and knowledge-based modelling. Biosci. Rep. 36, e00285 (2015)

    Article  CAS  PubMed  Google Scholar 

  25. Wootten, D. et al. The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism. Cell 165, 1632–1643 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Houssami, S. et al. Divergent structural requirements exist for calcitonin receptor binding specificity and adenylate cyclase activation. Mol. Pharmacol. 47, 798–809 (1995)

    CAS  PubMed  Google Scholar 

  27. Meadows, R. P., Nikonowicz, E. P., Jones, C. R., Bastian, J. W. & Gorenstein, D. G. Two-dimensional NMR and structure determination of salmon calcitonin in methanol. Biochemistry 30, 1247–1254 (1991)

    CAS  PubMed  Article  Google Scholar 

  28. Feyen, J. H. et al. N-terminal truncation of salmon calcitonin leads to calcitonin antagonists. Structure activity relationship of N-terminally truncated salmon calcitonin fragments in vitro and in vivo. Biochem. Biophys. Res. Commun. 187, 8–13 (1992)

    CAS  PubMed  Article  Google Scholar 

  29. Wootten, D., Simms, J., Miller, L. J., Christopoulos, A. & Sexton, P. M. Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations. Proc. Natl Acad. Sci. USA 110, 5211–5216 (2013)

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  30. Bailey, R. J. & Hay, D. L. Agonist-dependent consequences of proline to alanine substitution in the transmembrane helices of the calcitonin receptor. Br. J. Pharmacol . 151, 678–687 (2007)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Conner, A. C. et al. A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: implications for family B G-protein-coupled receptors. Mol. Pharmacol. 67, 20–31 (2005)

    CAS  PubMed  Article  Google Scholar 

  32. Koth, C. M. et al. Molecular basis for negative regulation of the glucagon receptor. Proc. Natl Acad. Sci. USA 109, 14393–14398 (2012)

    CAS  PubMed  Article  ADS  PubMed Central  Google Scholar 

  33. Mukund, S. et al. Inhibitory mechanism of an allosteric antibody targeting the glucagon receptor. J. Biol. Chem. 288, 36168–36178 (2013)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Yin, Y. et al. An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain. Cell Discov . 2, 16042 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Zhao, L. H. et al. Differential requirement of the extracellular domain in activation of class B G protein-coupled receptors. J. Biol. Chem. 291, 15119–15130 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Vohra, S. et al. Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies. J. R. Soc. Interface 10, 20120846 (2012)

    PubMed  Article  CAS  Google Scholar 

  37. Wootten, D. et al. A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures. Mol. Pharmacol. 89, 335–347 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Wootten, D. et al. Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor. Biochem. Pharmacol. 118, 68–87 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Conner, M. et al. Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR. Biochemistry 47, 8434–8444 (2008)

    CAS  PubMed  Article  Google Scholar 

  40. Furness, S. G., Wootten, D., Christopoulos, A. & Sexton, P. M. Consequences of splice variation on Secretin family G protein-coupled receptor function. Br. J. Pharmacol . 166, 98–109 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Harikumar, K. G., Ball, A. M., Sexton, P. M. & Miller, L. J. Importance of lipid-exposed residues in transmembrane segment four for family B calcitonin receptor homo-dimerization. Regul. Pept. 164, 113–119 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Harikumar, K. G. et al. Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery. Proc. Natl Acad. Sci. USA 109, 18607–18612 (2012)

    CAS  PubMed  Article  ADS  PubMed Central  Google Scholar 

  43. Black, J. W. & Leff, P. Operational models of pharmacological agonism. Proc. R. Soc. Lond. B Biol. Sci. 220, 141–162 (1983)

    CAS  PubMed  Article  ADS  Google Scholar 

  44. Peisley, A. & Skiniotis, G. 2D projection analysis of GPCR complexes by negative stain electron microscopy. Methods Mol. Biol . 1335, 29–38 (2015)

    PubMed  Article  Google Scholar 

  45. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005)

    PubMed  Article  Google Scholar 

  46. Shalev-Benami, M. et al. 2.8-Å cryo-EM structure of the large ribosomal subunit from the eukaryotic parasite Leishmania. Cell Reports 16, 288–294 (2016)

    CAS  PubMed  Article  Google Scholar 

  47. Zheng, S., Palovcak, E., Armache, J. P., Cheng, Y. & Agard, D. Anisotropic correction of beam-induced motion for improved single-particle electron cryo-microscopy. Preprint at https://doi.org/10.1101/061960 (2016)

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

    PubMed  PubMed Central  Article  Google Scholar 

  49. Penczek, P. A., Grassucci, R. A. & Frank, J. The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles. Ultramicroscopy 53, 251–270 (1994)

    CAS  PubMed  Article  Google Scholar 

  50. Yang, J. & Zhang, Y. Protein structure and function prediction using I-TASSER. Curr. Protoc. Bioinformatics 52, 1–15 (2015)

    Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Munk, C. et al. GPCRdb: the G protein-coupled receptor database - an introduction. Br. J. Pharmacol. 173, 2195–2207 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. Koole, C. et al. Polymorphism and ligand dependent changes in human glucagon-like peptide-1 receptor (GLP-1R) function: allosteric rescue of loss of function mutation. Mol. Pharmacol. 80, 486–497 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. Koole, C. et al. Allosteric ligands of the glucagon-like peptide 1 receptor (GLP-1R) differentially modulate endogenous and exogenous peptide responses in a pathway-selective manner: implications for drug screening. Mol. Pharmacol. 78, 456–465 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

This work was funded by the National Health and Medical Research Council of Australia (NHMRC) (grant numbers 1055134, 1061044 and 1120919) and NIH grants DK090165, NS092695. P.M.S. and A.C. are NHMRC Principal and Senior Principal Research Fellows, respectively. D.W. is a NHMRC Career Development Fellow. Computational studies were partially supported by Melbourne Bioinformatics at the University of Melbourne, grant number VR0024. Negative-stain imaging and cryo-EM screening was performed at the Monash Ramaciotti Centre for Cryo-Electron Microscopy. The GPCRdb (http://gpcrdb.org) was used for generation of initial alignments of human class B GPCR sequences. The authors thank the late M. Azria for 100 mg of sCT, used in initial work.

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Authors and Affiliations

Authors

Contributions

Y.-L.L. developed the expression and purification strategy (with D.M.T., S.G.B.F., B.K.K., D.W., G.S. and P.M.S.), performed virus production, insect cell expression, purification, complex stability, negative-stain EM, data acquisition/analysis, prepared samples for cryo-EM and assisted with manuscript preparation. M.K. performed phase-plate imaging, data collection, EM data processing and analysis and assisted with manuscript preparation. M.R. assisted with negative-stain analysis and conception of cryo-imaging by Volta phase plate, performed cryo-sample preparation, preliminary screening imaging and analysis and assisted with manuscript preparation. Y.Z. calculated the cryo-EM map, performed model building and refinement, and contributed to manuscript preparation. A.G. performed GTPγS and radioligand binding, model building and refinement and contributed to manuscript preparation. J.T. assisted in negative-stain EM screening. D.M.T. contributed to purification strategy, model refinement and manuscript preparation. S.G.B.F. provided project strategy and protein purification advice, assisted data interpretation and manuscript preparation. G.C. performed cloning for baculovirus expression. T.C. performed homology modelling, assisted with model refinement and manuscript preparation. R.D. developed Volta phase-plate cryo-EM data acquisition strategy and wrote automation scripts. W.B. organized and managed the Volta phase plate development project. L.J.M. provided insights into class B GPCRs, assisted with data interpretation and reviewed the manuscript. A.C. assisted with data interpretation and manuscript preparation. B.K.K. provided advice on CTR–Gs complex formation and purification. D.W. was responsible for overall project strategy and management (along with P.M.S.) and performed pharmacological characterization, interpreted data and wrote the manuscript. G.S. provided feedback to guide cryo-EM, oversaw EM data processing, structure determination and refinement, data interpretation and manuscript writing. P.M.S. was responsible for overall project strategy and management, data interpretation and writing the manuscript.

Corresponding authors

Correspondence to Denise Wootten, Georgios Skiniotis or Patrick M. Sexton.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks J. Standfuss and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Schematic of the CTR used in the study.

In our construct HA–Flag–3C–CTR–3C–8×His, the native signal peptide of the CTR (residues 2–24) was replaced with a HA signal peptide (red), Flag epitope (green) and a 3C cleavage site (yellow). The C terminus was modified with a 3C cleavage site (yellow) and a His epitope (blue). Also highlighted on the schematic are consensus glycosylation sites (purple) and class B GPCR conserved disulfide bonds. Residues highlighted in bold are the most conserved residue in each helix and represent residues x.50 for each helix according to the class B GPCR numbering. The location of the 16 amino acid insertion within ICL1 for a common splice variant of the CTR (CTRb) is shown. In addition, the locations of the truncation sites within the CTR C terminus/helix 8 assessed in this study are also highlighted.

Extended Data Figure 2 Pharmacology of the CTR construct used in this study.

ad, Pharmacological assessment in mammalian COS-7 cells (a, b) and HiveFive insect cells (c, d) of the untagged CTR and the construct shown in Extended Data Fig. 1 (HA–Flag–3C–CTR–3C–8×His). The presence of purification tags does not alter receptor pharmacology. a, Radioligand competition binding for sCT in competition with the radiolabelled ligand [125I]sCT(8–32) in whole cells transiently expressing wild-type CTR or HA–Flag–3C–CTR–3C–8×His. Data are normalized to maximum [125I]sCT(8–32) with nonspecific measured in the presence of 1μM unlabelled sCT(8–32). b, Concentration response curves assessing Gs activation via measurement of cAMP accumulation at wild-type CTR and HA–Flag–3C–CTR–3C–8×His in the presence of sCT. c, Radioligand competition binding for sCT or the radiolabelled ligand [125I]sCT(8–32) performed with HA–Flag–3C–CTR–3C–8×His in the presence of Gs protein heterotrimer reveals similar affinities in insect cells versus mammalian cells. The presence of Nb35 does not alter ligand affinity. d, Concentration response curves to assess G-protein activation by HA–Flag–3C–CTR–3C–8×His via GTPγS binding in the absence and presence of Gs protein heterotrimer reveals that the tagged CTR can robustly activate Gs in insect cells. e, GTPγS binding to HA–Flag–3C–CTR–3C–8×His in the presence of 1 μM sCT is inhibited by increasing concentrations of Nb35. All data are mean + s.e.m. of four independent experiments, conducted in duplicate or triplicate.

Extended Data Figure 3 Expression and purification of the sCT–CTR–Gs complex.

a, Flow chart of the purification steps for the human CTR (hCTR)–Gs complex. b, SDS–PAGE/western blot of samples obtained at various stages of hCTR–Gs purification. hCTR and the Gs heterotrimer were co-expressed in insect cell membrane. Addition of the agonist salmon calcitonin initiates complex formation and was solubilized by detergent. Solubilized hCTR and the hCTR–Gs complex were immobilized on Flag antibody resin. Flag-eluted fractions were further purified by SEC. An anti-His antibody was used to detect Flag–CTR–His, Gβ–His and Nb35–His (red) and an anti-Gs antibody was used to detect Gαs (green). c, Representative elution profile of Flag-purified complex on Superdex 200 Increase 10/30 SEC (top). SEC fractions containing hCTR–Gs complex (within dashed lines) were pooled, concentrated and analysed by SEC on Superose 6 Increase 10/30 column (bottom). d, SDS–PAGE/Coomassie blue stain of the purified complex concentrated from the Superose 6 Increase 10/30 column. e, The stability of the purified hCTR–Gs was monitored by SEC following incubation at 4 °C for 5 days. All images and SEC profiles are representative of more than ten experiments, except for e, which was performed once.

Extended Data Figure 4 Cryo-EM of the sCT–CTR–Gs complex.

a, Representative Volta phase plate (of 2,780 recordings) cryo-EM micrograph of the sCT–CTR–Gs complex (scale bar, 15 nm). b, Reference-free two-dimensional averages of the complex in maltose-neopentyl glycol/cholesterol hemisuccinate micelle. c, Gold-standard Fourier shell correlation (FSC) curves, showing the overall nominal resolution at 4.1 Å and 3.8 Å on the stable region including the transmembrane domain and Gs protein complex without AHD. d, Final three-dimensional density map coloured according to local resolution. e, FSC curves of the final refined model versus the final cryo-EM map (full dataset, black), of the outcome of model refinement with a half map versus the same map (red), and of the outcome of model refinement with a half map versus the other half map (green). At FSC = 0.5, the resolution is 4.1 Å. f, EM density of TM1, TM5, TM6, TM7 and helix 8.

Extended Data Figure 5 Flexibility of ECD and AHD in the sCT–CTR–Gs complex.

Representative maps from three-dimensional classification showing the dynamics of the CTR ECD and Gαs AHD. The overlaid maps are shown from top and side views. In the right panel the blue, green, purple and red density maps show the four 3D classifications. These are overlayed on the left to demonstrate the observed flexibility of the Gαs AHD and the CTR ECD.

Extended Data Figure 6 The N-terminal ECD of the CTR.

a, Rigid body fitting of the structure of CTR ECD bound to sCT (PDB: 5II0)22 into the corresponding regions of the cryo-EM map revealed additional density (close to residue 130) that may be attributed to glycosylation. bd, Asp mutation of four consensus glycosylation residues (N28D, N73D, N125D and N130D) reveals the relative unimportance of glycosylation on cell-surface expression (b), determined via cell-surface ELISA for the N-terminal epitope tag. c, Competition radioligand binding studies for sCT in competition with the radiolabelled ligand [125I]sCT(8–32) revealed reduced affinity for N130D, and to a lesser extent N125D, compared to the wild-type CTR. d, Concentration response curves for cAMP accumulation for mutant receptors relative to wild type show that N130D, and to a lesser extent N125D, reduce the potency of sCT in functional experiments. All data are mean + s.e.m. of five independent experiments, conducted in duplicate or triplicate.

Extended Data Figure 7 Molecular modelling of sCT peptide reveals potential interactions between peptide and receptor.

Cryo-EM density is shown in yellow fill, the sCT peptide model in yellow cartoon and the CTR in blue cartoon. a, Gln14 in sCT is predicted to form interactions with the backbone of ECL2. b, Ser5 and Thr6 are predicted to form hydrogen bonds with His302 in TM5 of the CTR, while Leu4 points down into the bundle towards TM6. c, Mutation of H302 to Ala (H302A) results in reduced potency for sCT in cAMP production (left) and phosphorylation of ERK1/2 (right) when expressed in 3T3-FlpIn cells. This supports a role H302 in sCT affinity. Data are mean + s.e.m. of four independent experiments performed in duplicate

Extended Data Figure 8 Comparisons of an inactive CTR homology model and the activate CTR structure.

a, Side view of the activate sCT–CTR–Gs complex transmembrane structure (blue) relative to the inactive CTR homology model (red). b, Tube representation for transmembrane domains showing extracellular (top) and cytoplasmic (bottom) views of the activate sCT–CTR–Gs complex transmembrane structure (blue) relative to the inactive CTR homology model (red). In a and b large differences are observed at the extracellular ends of TM6 and TM7, with additional differences within TM1 and TM5. In addition, a very large outward movement is observed within TM6 of the active structure relative to the inactive homology model at the intracellular face. c, The positions of class B conserved polar residues located within the inactive CTR homology model.

Extended Data Figure 9 CTR-Gs protein interactions.

a, The α5-helix of Gαs (orange) docks into a cavity formed on the intracellular side of the receptor (blue) by the opening of TM6. G-protein side chains within this cavity are supported by the cryo-EM map. b, Helix 8 of the CTR forms an amphipathic helix with multiple bulky aromatics heavily embedded within the detergent micelle that are evident in the map. Residues within the more polar face of helix 8 are in the vicinity of Gβ, where they probably form polar interactions, although specific side chain density in this region is not evident. c, ICL1 is located in close proximity to the G protein. A common CTR splice variant contains a 16 amino acid insertion within this loop (between Arg174 and Ser175), an insertion that would sterically hinder G-protein interactions with the receptor.

Extended Data Figure 10 Comparison of the activated β2AR and CTR viewed from the extracellular face.

Tube representation of the transmembrane domains of the CTR (blue) and β2AR (green) viewed from the cytoplasmic face (based on overlay of the Gs protein from each structure). Despite similarities in the position of transmembrane tips at the intracellular face, there are substantial differences in the location of the extracellular transmembrane tips, highlighting marked differences in the ligand binding mode and initiation of receptor activation between class A and B GPCRs.

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Liang, YL., Khoshouei, M., Radjainia, M. et al. Phase-plate cryo-EM structure of a class B GPCR–G-protein complex. Nature 546, 118–123 (2017). https://doi.org/10.1038/nature22327

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