Abstract

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

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Accessions

Data deposits

Coordinates and structure factors for the active M2 receptor in complex with Nb9-8 and iperoxo are deposited in the Protein Data Bank under accession code 4MQS, and the coordinates and structure factors of the same complex bound additionally to the allosteric modulator LY2119620 are deposited under accession code 4MQT.

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Acknowledgements

We acknowledge support from the National Science Foundation (graduate fellowship to A.C.K., and Award 1223785 to B.K.K.), the Stanford Medical Scientist Training Program (A.M. and A.M.R.), the American Heart Association (A.M.), the Ruth L. Kirschstein National Research Service Award (A.M.R.), National Institutes of Health grants NS02847123 and GM08311806 (B.K.K.), the Mathers Foundation (B.K.K., W.I.W. and K.C.G.), the Deutsche Forschungsgemeinschaft for the grant GM 13/10-1 (K.E., H.H., P.G.), the National Health and Medical Research Council (NHMRC) of Australia program grant 519461 (P.M.S. and A.C.), NHMRC Principal Research Fellowships (P.M.S. and A.C.), and the Howard Hughes Medical Institute (K.C.G.). This work was supported in part by the Intramural Research Program, NIDDK, NIH, US Department of Health and Human Services (J.H., K.H. and J.W.). We thank K. Leach for performing ERK assays, and B. Davie and P. Scammells for synthesis of iperoxo. We thank H. Xiao, C. H. Croy and D. A. Schober for functional characterization of LY2119620. We thank T. S. Kobilka for preparation of affinity chromatography reagents and F. S. Thian for help with cell culture.

Author information

Author notes

    • Andrew C. Kruse
    •  & Aaron M. Ring

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA

    • Andrew C. Kruse
    • , Aaron M. Ring
    • , Aashish Manglik
    • , William I. Weis
    • , K. Christopher Garcia
    •  & Brian K. Kobilka
  2. Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, California 94305, USA

    • Aaron M. Ring
    • , William I. Weis
    •  & K. Christopher Garcia
  3. Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA

    • Jianxin Hu
    • , Kelly Hu
    •  & Jürgen Wess
  4. Department of Chemistry and Pharmacy, Friedrich Alexander University, Schuhstrasse 19, 91052 Erlangen, Germany

    • Katrin Eitel
    • , Harald Hübner
    •  & Peter Gmeiner
  5. Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

    • Els Pardon
    •  & Jan Steyaert
  6. Structural Biology Research Centre, VIB, Pleinlaan 2, B-1050 Brussels, Belgium

    • Els Pardon
    •  & Jan Steyaert
  7. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia

    • Celine Valant
    • , Patrick M. Sexton
    •  & Arthur Christopoulos
  8. Neuroscience, Eli Lilly & Co., Indianapolis, Indiana 46285, USA

    • Christian C. Felder

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Contributions

A.C.K. expressed and purified M2 receptor for yeast display and crystallographic experiments, performed crystallization, data collection, and structure refinement, and performed radioligand binding assays to validate nanobody activity. A.C.K., A.M.R. and A.M. designed experiments to identify nanobodies by yeast display. A.M.R. performed all yeast selections, and expressed and purified Nb9-8 and other nanobodies. J.H. and K.H. performed site-directed mutagenesis and characterization of resulting mutants. K.E. synthesized FAUC123. H.H. performed cell assays and radioligand binding to characterize FAUC123. C.V. performed pharmacological characterization of LY2119620. P.M.S. and A.C. supervised pharmacological characterization of LY2119620. C.C.F. designed key solubility, physical chemistry and ligand analysis to select LY2119620 as an appropriate co-crystallization candidate for the M2 receptor. P.G. supervised synthesis and characterization of FAUC123. E.P. and J.S. performed llama immunization, cDNA production, and performed selections by phage display. W.I.W. supervised structure refinement. K.C.G. supervised yeast selection experiments. J.W. supervised mutagenesis experiments and analysed results. B.K.K. provided overall project supervision, and with A.C.K., A.M.R. and A.M. wrote the manuscript with assistance from A.C. and J.W.

Competing interests

A.C.K., A.M.R. and A.M. have applied for a patent on the yeast display and screening methods used to identify the conformationally selective nanobody used to obtain the crystal structure.

Corresponding author

Correspondence to Brian K. Kobilka.

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https://doi.org/10.1038/nature12735

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