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Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Nature volume 561, pages 492–497 (2018)Cite this article

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Abstract

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the Gs-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

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Fig. 1: The cryo-EM structure of CGRP–CLR–RAMP1–Gs reveals molecular details of the RAMP-receptor interface.
Fig. 2: RAMP1 forms stable interactions with the CLR core and ECD.
Fig. 3: The CGRP-binding site.
Fig. 4: The CTR and CGRP receptor complexes display similar backbone conformations but have distinct conformations of the Gαs-Ras domain.

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Data availability

All relevant data are available from the authors and/or included in the manuscript or Supplementary Information. Atomic coordinates and the cryo-EM density map have been deposited in the Protein Data Bank under accession number 6E3Y and the Electron Microscopy Data Bank, entry EMD-8978.

References

  1. Hay, D. L., Garelja, M. L., Poyner, D. R. & Walker, C. S. Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25. Br. J. Pharmacol. 175, 3–17 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Russell, F. A., King, R., Smillie, S. J., Kodji, X. & Brain, S. D. Calcitonin gene-related peptide: physiology and pathophysiology. Physiol. Rev. 94, 1099–1142 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dickerson, I. M., Bussey-Gaborski, R., Holt, J. C., Jordan, P. M. & Luebke, A. E. Maturation of suprathreshold auditory nerve activity involves cochlear CGRP-receptor complex formation. Physiol. Rep. 4, e12869 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Walker, C. S. et al. Mice lacking the neuropeptide α-calcitonin gene-related peptide are protected against diet-induced obesity. Endocrinology 151, 4257–4269 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Karsan, N. & Goadsby, P. J. Calcitonin gene-related peptide and migraine. Curr. Opin. Neurol. 28, 250–254 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. McLatchie, L. M. et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393, 333–339 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Barbash, S., Lorenzen, E., Persson, T., Huber, T. & Sakmar, T. P. GPCRs coevolved with receptor activity-modifying proteins, RAMPs. Proc. Natl Acad. Sci. USA 114, 12015–12020 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hay, D. L. & Pioszak, A. A. Receptor activity-modifying proteins (RAMPs): new insights and roles. Annu. Rev. Pharmacol. Toxicol. 56, 469–487 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Routledge, S. J., Ladds, G. & Poyner, D. R. The effects of RAMPs upon cell signalling. Mol. Cell. Endocrinol. 449, 12–20 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Wootten, D. et al. Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function. Br. J. Pharmacol. 168, 822–834 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Christopoulos, A. et al. Novel receptor partners and function of receptor activity-modifying proteins. J. Biol. Chem. 278, 3293–3297 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Booe, J. M., Warner, M. L., Roehrkasse, A. M. & Pioszak, A. A. Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor. Mol. Cell 58, 1040–1052 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Booe, J. M. et al. Probing the mechanism of receptor activity-modifying protein modulation of GPCR ligand selectivity through rational design of potent adrenomedullin and calcitonin gene-related peptide antagonists. Mol. Pharmacol. 93, 355–367 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liang, Y. L. et al. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature 555, 121–125 (2018).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. 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  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dal Maso, E. et al. Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy. Biochem. Pharmacol. 150, 214–244 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Liang, Y.-L. et al. Dominant negative G proteins enhance formation and purification of agonist-GPCR–G protein complexes for structure determination ACS Pharmacol Transl Sci https://doi.org/10.1021/acsptsci.8b00017 (2018).

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Khoshouei, M., Radjainia, M., Baumeister, W. & Danev, R. Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate. Nat. Commun. 8, 16099 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ter Haar, E. et al. Crystal structure of the ectodomain complex of the CGRP receptor, a class-B GPCR, reveals the site of drug antagonism. Structure 18, 1083–1093 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Watkins, H. A. et al. Receptor activity-modifying proteins 2 and 3 generate adrenomedullin receptor subtypes with distinct molecular properties. J. Biol. Chem. 291, 11657–11675 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Woolley, M. J. et al. Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs. Biochem. Pharmacol. 142, 96–110 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Weston, C. et al. Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors. J. Biol. Chem. 291, 21925–21944 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bower, R. L. et al. Molecular signature for receptor engagement in the metabolic peptide hormone amylin. ACS Pharmacol. Transl. Sci. https://doi.org/10.1021/acsptsci.8b00002 (2018).

    Article  CAS  Google Scholar 

  28. Woolley, M. J. et al. Understanding the molecular functions of the second extracellular loop (ECL2) of the calcitonin gene-related peptide (CGRP) receptor using a comprehensive mutagenesis approach. Mol. Cell. Endocrinol. 454, 39–49 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. Hay, D. L. et al. Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation. Br. J. Pharmacol. 171, 415–426 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. Barwell, J., Conner, A. & Poyner, D. R. Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim. Biophys. Acta 1813, 1906–1916 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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).

    Article  CAS  PubMed  Google Scholar 

  32. Woolley, M. J. et al. The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach. J. R. Soc. Interface 10, 20130589 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Conner, A. C., Simms, J., Howitt, S. G., Wheatley, M. & Poyner, D. R. The second intracellular loop of the calcitonin gene-related peptide receptor provides molecular determinants for signal transduction and cell surface expression. J. Biol. Chem. 281, 1644–1651 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Simms, J. et al. Structure-function analysis of RAMP1 by alanine mutagenesis. Biochemistry 48, 198–205 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Zhang, H. et al. Structure of the glucagon receptor in complex with a glucagon analogue. Nature 553, 106–110 (2018).

    Article  ADS  CAS  PubMed  Google Scholar 

  36. Simms, J. et al. Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex. Biochemistry 57, 4915–4922 (2018).

    Article  CAS  PubMed  Google Scholar 

  37. Garelja, M. L. et al. Receptor activity modifying proteins have limited effects on the Class B G protein-coupled receptor calcitonin receptor-like receptor stalk. Biochemistry 57, 1410–1422 (2018).

    Article  CAS  PubMed  Google Scholar 

  38. Hay, D. L., Christopoulos, G., Christopoulos, A. & Sexton, P. M. Determinants of 1-piperidinecarboxamide, N-[2-[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS) affinity for calcitonin gene-related peptide and amylin receptors–the role of receptor activity modifying protein 1. Mol. Pharmacol. 70, 1984–1991 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Abagyan, R. & Totrov, M. Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. J. Mol. Biol. 235, 983–1002 (1994).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  44. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 3651 (2005).

    Article  Google Scholar 

  45. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Zheng, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  ADS  CAS  Google Scholar 

  47. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hager, M. V., Clydesdale, L., Gellman, S. H., Sexton, P. M. & Wootten, D. Characterization of signal bias at the GLP-1 receptor induced by backbone modification of GLP-1. Biochem. Pharmacol. 136, 99–108 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Jacobson, M. P. et al. A hierarchical approach to all-atom protein loop prediction. Proteins 55, 351–367 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Goldfeld, D. A., Zhu, K., Beuming, T. & Friesner, R. A. Successful prediction of the intra- and extracellular loops of four G-protein-coupled receptors. Proc. Natl Acad. Sci. USA 108, 8275–8280 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. Eswar, N. et al. Comparative protein structure modeling using Modeller. Curr. Protoc. Bioinformatics 15, 5.6.1– 5.6.30 (2006).

    Article  Google Scholar 

  53. Huang, J. & MacKerell, A. D. CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J. Comput. Chem. 34, 2135–2145 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Doerr, S., Harvey, M. J., Noé, F. & De Fabritiis, G. HTMD: high-throughput molecular dynamics for molecular discovery. J. Chem. Theory Comput. 12, 1845–1852 (2016).

    Article  CAS  PubMed  Google Scholar 

  55. Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: an automated pipeline for the setup of Poisson–Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–667 (2004).

    Article  CAS  Google Scholar 

  56. Olsson, M. H. M., Søndergaard, C. R., Rostkowski, M. & Jensen, J. H. PROPKA3: consistent treatment of internal and surface residues in empirical pK predictions. J. Chem. Theory Comput. 7, 525–537 (2011).

    Article  CAS  PubMed  Google Scholar 

  57. Sommer, B. Membrane packing problems: a short review on computational membrane modeling methods and tools. Comput. Struct. Biotechnol. J. 5, e201302014 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Lomize, M. A., Lomize, A. L., Pogozheva, I. D. & Mosberg, H. I. OPM: orientations of proteins in membranes database. Bioinformatics 22, 623–625 (2006).

    Article  CAS  PubMed  Google Scholar 

  59. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926 (1983).

    Article  ADS  CAS  Google Scholar 

  60. Harvey, M. J., Giupponi, G. & Fabritiis, G. D. ACEMD: accelerating biomolecular dynamics in the microsecond time scale. J. Chem. Theory Comput. 5, 1632–1639 (2009).

    Article  CAS  PubMed  Google Scholar 

  61. Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684 (1984).

    Article  ADS  CAS  Google Scholar 

  62. Loncharich, R. J., Brooks, B. R. & Pastor, R. W. Langevin dynamics of peptides: the frictional dependence of isomerization rates of N-acetylalanyl-N′-methylamide. Biopolymers 32, 523–535 (1992).

    Article  CAS  PubMed  Google Scholar 

  63. Kräutler, V., van Gunsteren, W. F. & Hünenberger, P. H. A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J. Comput. Chem. 22, 501–508 (2001).

    Article  Google Scholar 

  64. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577 (1995).

    Article  ADS  CAS  Google Scholar 

  65. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The work was supported by the Monash University Ramaciotti Centre for Cryo-Electron Microscopy, National Health and Medical Research Council of Australia (NHMRC) project grant (1120919), and NHMRC program grant (1055134). P.M.S. and A.C. are NHMRC Principal and Senior Principal Research Fellows, respectively. D.W. is a NHMRC Career Development Fellow, and C.K. is a NHMRC CJ Martin Fellow. A.G. is an Australian Research Council DECRA Fellow. D.L.H. is a James Cook Research Fellow and is supported by the Marsden Fund (both Royal Society of New Zealand). C.A.R. is a Royal Society Industry Fellow and acknowledges support from the BBSRC (BB/M006883/1). We are grateful to G. Christopoulos and V. Julita for assay and technical support, T. Coudrat for initial homology modelling of CLR from the active CTR, and to S. Furness, P. Zhao and D. Thal for useful discussion.

Reviewer information

Nature thanks K. Caron, S, Dang, B. Wu and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. Maryam Khoshouei

    Present address: Novartis Institutes for Biomedical Research, Novartis Pharma, Basel, Switzerland

  2. These authors contributed equally: Yi-Lynn Liang, Maryam Khoshouei

Authors and Affiliations

  1. Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    Yi-Lynn Liang, Alisa Glukhova, Cassandra Koole, Mazdak Radjainia, Laurence J. Miller, Arthur Christopoulos, Denise Wootten & Patrick M. Sexton

  2. Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany

    Maryam Khoshouei, Jürgen M. Plitzko & Wolfgang Baumeister

  3. School of Biological Sciences, University of Essex, Colchester, UK

    Giuseppe Deganutti & Christopher A. Reynolds

  4. CSIRO Biomedical Manufacturing, Melbourne, Victoria, Australia

    Thomas S. Peat

  5. Thermo Fisher Scientific, Eindhoven, The Netherlands

    Mazdak Radjainia

  6. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, USA

    Laurence J. Miller

  7. School of Biological Sciences, University of Auckland, Auckland, New Zealand

    Deborah L. Hay

  8. Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand

    Deborah L. Hay

  9. School of Pharmacy, Fudan University, Shanghai, China

    Denise Wootten & Patrick M. Sexton

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Contributions

Y.-L.L performed virus production, insect cell expression, purification, negative stain electron microscopy, data acquisition and analysis, and prepared samples for cryo-EM, was responsible for model building and refinement, and assisted with manuscript preparation. M.K. performed cryo-sample preparation, phase-plate imaging, data collection, electron microscopy data processing and analysis, calculated the cryo-EM map and assisted with manuscript preparation. G.D. performed molecular dynamics simulations and assisted in manuscript preparation. A.G. assisted with model building and refinement and contributed to manuscript preparation. T.S.P. assisted with model building and refinement and reviewed the manuscript. C.K. performed cell-based assays and data analysis and reviewed the manuscript. M.R. performed preliminary cryo-EM screening and reviewed the manuscript. J.M.P. and W.B. organized and managed the Volta phase-plate development project. D.L.H. provided insights into the CGRP receptor, assisted with data interpretation, and reviewed the manuscript. 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. C.A.R. designed molecular dynamics simulations, assisted in data interpretation and contributed to writing of the manuscript. D.W. was responsible for overall project strategy and management, data analysis and interpretation and contributed to writing of the manuscript. P.M.S. was responsible for overall project strategy and management, data interpretation and wrote the manuscript.

Corresponding authors

Correspondence to Denise Wootten or Patrick M. Sexton.

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Extended data figures and tables

Extended Data Fig. 1 Amino acid sequences of the CGRP peptide, CLR and RAMP1 constructs used for determination of structure.

The sequences are annotated to denote the location of the haemagglutinin (HA) signal sequence (red highlight), 3C cleavage sites (grey highlight), Flag (dark olive-green highlight) and His tags (purple highlight). The substituted sequences of the native proteins are listed above the construct sequences and highlighted in blue. Transmembrane helical domains in CLR and RAMP1 are boxed and highlighted in green. Segments of the proteins that were not resolved in the cryo-EM map are highlighted in yellow. Amino acids for which backbone density was present but there was limited side-chain density were stubbed in the model; these are shown in bold red in the sequences.

Extended Data Fig. 2 CGRP receptor pharmacology and purification of the CGRP–CLR–RAMP1–Gs complex.

a, Pharmacology of untagged CLR–RAMP1 (wild-type (WT) CLR–RAMP1) and the purification construct (HA–Flag–CLR and Flag–RAMP1), in CGRP-mediated cAMP accumulation assays performed in transiently transfected COS-7 cells. n = 5 independent experiments with triplicate repeats; data are mean + s.e.m. b, Expression and purification strategy. c, Final size-exclusion chromatography elution profile of the complex. d, SDS–PAGE and Coomassie blue staining of the size-exclusion chromatography peak, demonstrating the presence of each of the components of the complex.

Extended Data Fig. 3 Volta phase-plate imaging of the CGRP–CLR–RAMP1–Gs complex.

a, Volta phase-plate micrograph of the complex (representative of 3,180 movies). High-contrast phase-plate imaging facilitates robust particle selection despite low defocus and tight packing of particles. b, RELION 2D class averages. c, Workflow for map refinement. d, Final 3D cryo-EM map calculated in RELION after auto-refinement and map sharpening. e, Gold standard Fourier shell correlation curve; the overall nominal resolution is 3.26 Å. f, Model overfitting was evaluated by randomly displacing all atoms by 0.5 Å and refined against one cryo-EM half map. Fourier shell correlation curves were calculated between the resulting model and the half map used for refinement (green); the resulting model and the other half map for cross validation (red), and the final refined model and the full map (blue). g, Potential lipid interaction with the base of TM4 and TM2 of CLR.

Extended Data Fig. 4 Atomic-resolution model of the CGRP–CLR–RAMP1–Gs complex in the cryo-EM density map.

Cryo-EM density map and model are shown for all seven transmembrane helices and H8 of the receptor, the CGRP peptide (excluding the Lys24-Asn25-Asn26 sequence that was not resolved in the map), the RAMP transmembrane domain and each of the RAMP ECD helices. There was only limited side-chain density for RAMP1 H1, with side chains modelled from rigid-body fitting of the RAMP1 ECD in PDB: 4RWG12. The N-terminal (αH1) and C-terminal (αH5) α-helices of the Gαs-Ras domain are also shown. Superscript P indicates residues of CGRP.

Extended Data Fig. 5 Alignment of modelled active complex and X-ray structure.

Backbone of the ECD of CLR (blue ribbon) and RAMP1 (orange ribbon) from the modelled, active complex, and the structure of the isolated CLR–RAMP1 ECD complex solved by X-ray crystallography12 (light grey ribbon). The structures were aligned on the RAMP1 ECD. The CLR loops (loops 1–5) are annotated. The CLR loop 1 and loop 5 sequences that were not resolved in the cryo-EM map are indicated by dotted black arrows. Differences in the backbone position of CLR loops 4 and 5 are indicated in blue (active complex) and grey (isolated ECD complex) dotted arrows. The location of the CGRP peptide is shown in dark red.

Extended Data Fig. 6 RMSF for CGRP and CLR taken from the three simulations.

Simulations of CLR–CGRP–RAMP1–Gαβγ–Nb35 (black, 2.4 μs), CLR–CGRP–RAMP1–Gα(371–394) (purple, 2 μs) and CLR–CGRP–Gα(371–394) (blue, 2 μs). a, The CLR ECD region. b, The CLR transmembrane region. c, CGRP (superposed on T6–S17, and therefore valid for the N-terminal half). In general, the missing segments in the cryo-EM density map correspond to regions of high RMSF, and indeed the difficulty of fitting the ECD as a whole is linked to its high RMSF (a; Supplementary Videos 2, 3). The segments missing from the ECD (D55ECD–V63ECD) and (Q107ECD–G109ECD) correspond to external loop regions furthest removed from the transmembrane domain. Despite their polar nature they displayed no persistent interactions during the molecular dynamics simulations; D55ECD–V63ECD displayed the largest backbone RMSF of 8 Å, whereas Q107ECD–G109ECD displayed a similarly high RMSF of 7.5 Å. The next-highest RMSF peaks around A79ECD–G81ECD and P115ECD–S117ECD are just a little lower, but are nonetheless resolved (a). Within the transmembrane domain, ICL3 (H324ICL3–S328ICL3) and ECL3 (P356ECL3–E362ECL3) both contain missing residues and have a high RMSF above 4.5 Å (b). This region displays no persistent interactions during the molecular dynamics simulations, although CGRP does interact with the proximal (non-missing) region of ECL3. The high RMSF values for ICL1 (3.6 Å) and ICL2 (3.6 Å) give rise to stubbed residues (K1672.40) and E248ICL2–Q250ICL2) but the backbone is resolved. For CGRP, the peak in the RMSF around residue 26 (c) corresponds to the three highly mobile external residues (Lys24-Asn25-Asn26) in the outward-facing loop that do not interact with CLR (Extended Data Fig. 8); these residues could not be placed from the electron density. These three CGRP residues form a hinge, enabling changes in the orientation of the CLR ECD, especially in the absence of RAMP1; the higher RMSF values C-terminal to this are an artefact of the superposition strategy and the two-domain nature of CLR, but their relative values still hold. The high mobility of some of the extracellular loops is visible in videos (Supplementary Videos 1–3).

Extended Data Fig. 7 RAMP1 makes extensive stable interactions with CLR.

a, Hydrogen bonds between RAMP1 and CLR during molecular dynamics simulations (6.4 μs). The total persistence is plotted onto the experimental structure according to a rainbow colour scale, with residues that are never involved in dark blue and residues that are highly involved in red. The receptor is shown as a bulky ribbon, RAMP1 as a thin coloured ribbon and the peptide as a thin white ribbon. Key side chains are shown, but for intermittent hydrogen bonds the rotameric state has been modified to show an interaction. Residues forming an interaction network are labelled with the same colour. Left, overall topology of the system. Right top, magnified view of the upper portion of the CLR transmembrane domain and ECD; right bottom, view rotated by 90° on the z axis. Hydrogen bonds involved in the RAMP1–CLR interaction, R112R–E47ECD and D113R–T288ECL2/H289ECL2 are notable because they link the transmembrane domain to the ECD, and for stabilizing ECL2. Other hydrogen bonds implicated in stabilizing the CLR and RAMP1 ECD interaction include S107R–E47ECD, R102R–D55ECD, H97R–Q50ECD, D90R–Y49ECD, D71R–R38ECD and E29R–R119ECD. Quantitative data on the persistence of hydrogen bonds during the simulations are reported in Supplementary Table 2. b, Contacts between RAMP1 and CLR during simulations (6.4 μs). The total persistence of a residue side chain is plotted onto the experimental structure according to a cyan–maroon colour scale, with residues that are never involved in cyan and residues that are highly involved in maroon. The peptide (italics, dashed line) is depicted as a thin ribbon, whereas the receptor (solid line) is shown as a bulky ribbon and transparent surface. Left, overall topology of the system. Top right, the most-persistent interactions involving RAMP residues and the CLR ECD, W59R, I63R, Y66R, H97R and I106R help to anchor αH3 and the C-terminal RAMP1 regions of αH2 to (residues M42ECD, T43ECD, Y46ECD, Y49ECD, Q50ECD and M53ECD of the CLR ECD). Bottom right, the most-persistent hydrophobic interactions between the transmembrane domains of RAMP1 and CLR, namely I123R, P126R, T130R, T134R and V137R (plus S141R) help to anchor the RAMP transmembrane helix to CLR (TM3–TM5; CLR residues Y277ECL2, H289ECL2, A3005.45, I2353.52, F2624.52, L2584.48 and W2544.44).

Extended Data Fig. 8 Effect of alanine mutagenesis of CLR or RAMP1 on CGRP potency in cAMP accumulation assays.

a, ECD alanine mutations. b, CLR core alanine mutations. Residues that have been mutated are displayed in x-stick format. Mutated residues with no effect on signalling are coloured off-white. Residues that have significantly altered CGRP signalling12,23,28,30,31,32,34,37,38 are also highlighted in transparent CPK representation, coloured according to magnitude of effect. Yellow, <10 fold; dark orange, 10–100 fold; red, 100–1,000 fold; black, >1,000 fold. The backbones of CLR and RAMP (solid lines) are displayed in transparent, off-white coloured ribbon. The CGRP peptide (dashed lines) is represented in x-stick format with carbon atoms in dark red and polar atoms coloured in red or blue.

Extended Data Fig. 9 CGRP makes extensive stable interactions with CLR.

a–d, Distances between CGRP and CLR residues relevant to key hydrogen bonds. The x axis denotes sampling time for the 16 merged molecular dynamics replicas of the whole system (each replica is separated by a vertical dashed line). a, Distance between the peptide Asp3 carboxylic carbon and receptor R3556.59 guanidinium carbon. b, Distance between the peptide Thr6 side-chain oxygen atom and the receptor H2955.40 side-chain nitrogen atoms (for each frame, the closest nitrogen to Thr6 was considered). c, Distance between the peptide Arg11 guanidinium carbon and the receptor D3667.39 carboxylic carbon. d, Distance between peptide Arg18 guanidinium carbon and receptor D287ECL2 carboxylic carbon. In most cases, the distances corresponding to hydrogen-bond formation are slightly longer than the standard 2.8 Å. e, Hydrogen bonds between CGRP and CLR during molecular dynamics simulations (6.4 μs). The total persistence of a residue side chain is plotted onto the experimental structure according to a rainbow colour scale, with residues that are never involved in blue and residues that are highly involved in red. The peptide (italics, dashed line) is depicted as thin ribbon, whereas the receptor (solid line) is shown as bulky ribbon. Key side chains are shown, but for intermittent hydrogen bonds, the rotameric state has been modified to show an interaction. Residues forming an interaction network are labelled with the same colour. Bottom, hydrogen bonds between the CGRP N terminus and the transmembrane bundle of CLR. Top, hydrogen bonds between the CGRP C terminus and the ECD of CLR; quantitative data on the persistence of hydrogen bonds during the simulations are reported in Supplementary Table 3. f, Contacts between CGRP and CLR–RAMP1 during molecular dynamics simulations (6.4 μs). The total persistence of a residue side chain is plotted onto the experimental structure according to a cyan–maroon colour scale, with residues that are never involved in cyan and residues that are highly involved in maroon. The peptide (italics, dashed line) is depicted as a thin ribbon, while the receptor (solid line) is shown as a bulky ribbon and transparent surface. Left, contacts between the N terminus of CGRP and the transmembrane bundle of the CLR: highly persistent hydrophobic interactions characterize peptide residues Leu12, Leu16, His10 and receptor residues L1952.68, A1381.36 and H2955.40. Right, contacts between the C terminus of CGRP and the ECD of CLR; highly persistent contacts characterize peptide residues Val32, Thr30, Phe37 and receptor residues Q93ECD and W72ECD. RAMP1 residues F83R, W84R are mainly engaged by CGRP residue Phe37.

Extended Data Fig. 10 Class B GPCRs display similar active state conformations.

a, b, Alignment of the CGRP–CLR–RAMP1, sCT–CTR, ExP5–GLP-1R and GLP-1–GLP-1R structures (aligned on the transmembrane domains). Regions of divergence between CLR/CTR and GLP-1R are circled. In a, RAMP1 has been omitted for clarity. c, Position of the Gαs-Ras domain in the CTR (left), GLP-1R (GLP-1 bound; middle) and GLP-1R (ExP5 bound; right). The receptor transmembrane domains were aligned. Only the CLR (blue) and RAMP1 (orange) are displayed for clarity. d, The Gαs-Ras domain from each of the four structures, aligned to the Gαs-Ras of the CGRP receptor (CGRPR) complex.

Supplementary information

Supplementary Tables

This file contains Supplementary Tables 1-6.

Reporting Summary

Video 1

The CGRP (grey), CLR (green), RAMP1 (orange), G-protein (α subunit in blue, β subunit in red and γ subunit in yellow), Nb35 (maroon) complex simulated during a 400 ns long MD replica. Water molecules, ions and the lipid bilayer have been removed for clarity.

Video 2

Details of the extracellular TMs bundle during a 500 ns long MD replica, performed on the CGRP-CLR-RAMP1-G-protein complex. The hydrogen bonds formed between CGRP (orange), and CLR (cyan), and between CGRP (orange) and RAMP1 (green) are highlighted as dotted lines throughout the simulation.

Video 3

Comparison between two different 500 ns long MD simulations performed on: Left, CGRP (orange), CLR (green ribbon and transparent surface), RAMP1 (magenta ribbon and transparent surface), G-protein (371-394) complex. Right, CGRP (orange), CLR (green ribbon and transparent surface), G-protein (371-394) complex.

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Liang, YL., Khoshouei, M., Deganutti, G. et al. Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor. Nature 561, 492–497 (2018). https://doi.org/10.1038/s41586-018-0535-y

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