Abstract

Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases1,2,3,4,5,6,7,8. Previous work9,10,11 has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved12,13,14. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR–NNC0640–mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation—which requires conformational changes of the stalk, first extracellular loop and TMD—that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

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Acknowledgements

This work was supported by CAS Strategic Priority Research Program XDB08020000, CAS grants QYZDB-SSW-SMC024 (B.W.) and QYZDB-SSW-SMC054 (Q.Z.), the National Science Foundation of China grants 31422017 (B.W.) and 81525024 (Q.Z.), the Shanghai Science and Technology Development Fund 15DZ2291600 (M.-W.W.), the E-Institutes of Shanghai Municipal Education Commission (E09013), the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (second phase) under Grant No. U1501501, and the Canada Excellence Research Chairs program and the Canadian Institute for Advanced Research (O.P.E.). O.P.E. holds the Anne and Max Tanenbaum Chair in Neuroscience. We also thank the computer centre of East China Normal University for computational resources. The synchrotron radiation experiments were performed at the BL41XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (proposal numbers 2016B2517, 2016B2518, 2017A2505 and 2017A2506). We thank the beamline staff members K. Hasegawa, N. Mizuno, T. Kawamura and H. Murakami of the BL41XU for help with X-ray data collection.

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Affiliations

  1. CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China

    • Haonan Zhang
    • , Anna Qiao
    • , Dehua Yang
    • , Antao Dai
    • , Xiaoqing Cai
    • , Hui Zhang
    • , Cuiying Yi
    • , Ming-Wei Wang
    • , Hualiang Jiang
    • , Qiang Zhao
    •  & Beili Wu
  2. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China

    • Haonan Zhang
    • , Anna Qiao
    • , Hualiang Jiang
    •  & Qiang Zhao
  3. University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China

    • Haonan Zhang
    • , Anna Qiao
    • , Hui Zhang
    • , Can Cao
    • , Ming-Wei Wang
    • , Qiang Zhao
    •  & Beili Wu
  4. Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, 100 Science Avenue, Zhengzhou 450001, China

    • Linlin Yang
  5. Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Ned Van Eps
    •  & Oliver P. Ernst
  6. Novo Nordisk A/S, Novo Nordisk Park, Måløv 2760, Denmark

    • Klaus S. Frederiksen
    • , Jesper Lau
    •  & Steffen Reedtz-Runge
  7. The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Pudong, Shanghai 201203, China

    • Dehua Yang
    • , Antao Dai
    • , Xiaoqing Cai
    •  & Ming-Wei Wang
  8. National Laboratory of Biomacromolecules, National Center of Protein Science - Beijing, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

    • Can Cao
    •  & Lingli He
  9. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China

    • Huaiyu Yang
  10. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

    • Oliver P. Ernst
  11. GPCR Consortium, San Marcos, California 92078, USA

    • Michael A. Hanson
  12. iHuman Institute, ShanghaiTech University, 393 Hua Xia Zhong Road, Shanghai 201210, China

    • Raymond C. Stevens
  13. School of Life Science and Technology, ShanghaiTech University, 393 Hua Xia Zhong Road, Pudong, Shanghai 201210, China

    • Raymond C. Stevens
    • , Ming-Wei Wang
    •  & Beili Wu
  14. School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China

    • Ming-Wei Wang
  15. Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China

    • Hualiang Jiang
  16. CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences, Beijing 100101, China

    • Qiang Zhao
    •  & Beili Wu

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Contributions

Ha.Z. optimized the construct, developed the purification procedure and purified the GCGR proteins for crystallization, performed crystallization trials and optimized crystallization conditions. A.Q. helped with construct optimization and crystallization trials. L.Y. performed and analysed molecular dynamics simulations. N.V.E. performed and analysed DEER spectroscopy. K.S.F. performed and analysed binding and potency assays of glucagon and NNC1702. D.Y., A.D. and X.C. designed, performed and analysed the whole-cell glucagon binding assay. Hu.Z. collected the X-ray diffraction data. C.Y. expressed the GCGR proteins. C.C. and L.H. helped to analyse the conformational variety of GCGR. J.L., O.P.E., M.A.H., R.C.S, M.-W.W. and S.R.-R. helped with structure analysis and interpretation, and edited the manuscript. O.P.E. oversaw DEER spectroscopy. M.-W.W. oversaw the whole-cell glucagon binding assay. H.Y. and H.J. oversaw molecular dynamics simulations and commented on the manuscript. S.R.-R. designed the peptide and oversaw ligand characterization of NNC1702. Q.Z. and B.W. initiated the project, planned and analysed experiments, solved the structures, supervised the research and wrote the manuscript with input from all co-authors.

Competing interests

K.S.F., J.L. and S.R.-R. are employees of Novo Nordisk, a pharmaceutical company focused on class B GPCRs for type 2 diabetes. R.C.S. is a founder and board member of Bird Rock Bio, a company focused on GPCR therapeutic antibodies. The remaining authors declare no competing financial interests.

Corresponding authors

Correspondence to Qiang Zhao or Beili Wu.

Reviewer Information Nature thanks G. Schertler, D. Wootten and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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