Abstract

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β2 adrenergic receptor (β2AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β2AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for β2AR–Nb80 are deposited in the Protein Data Bank (accession code 3P0G).

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Acknowledgements

We acknowledge support from National Institutes of Health Grants NS028471 and GM083118 (B.K.K.), GM56169 (W.I.W.), P01 GM75913 (S.H.G), and P60DK-20572 (R.K.S.), the Mathers Foundation (B.K.K. and W.I.W.), the Lundbeck Foundation (Junior Group Leader Fellowship, S.G.F.R.), the University of Michigan Biomedical Sciences Scholars Program (R.K.S.), the Fund for Scientific Research of Flanders (FWO-Vlaanderen) and the Institute for the encouragement of Scientific Research and Innovation of Brussels (ISRIB) (E.P. and J.S.).

Author information

Author notes

    • Søren G. F. Rasmussen
    • , Hee-Jung Choi
    •  & Juan Jose Fung

    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

    • Søren G. F. Rasmussen
    • , Hee-Jung Choi
    • , Juan Jose Fung
    • , Daniel M. Rosenbaum
    • , Foon Sun Thian
    • , Tong Sun Kobilka
    • , William I. Weis
    •  & Brian K. Kobilka
  2. Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark

    • Søren G. F. Rasmussen
  3. Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, California 94305, USA

    • Hee-Jung Choi
    •  & William I. Weis
  4. Department of Molecular and Cellular Interactions, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel, B-1050 Brussels, Belgium

    • Els Pardon
    •  & Jan Steyaert
  5. Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium

    • Els Pardon
    •  & Jan Steyaert
  6. Boehringer Ingelheim Pharma GmbH & Co. KG, Germany

    • Paola Casarosa
    • , Andreas Schnapp
    • , Ingo Konetzki
    •  & Alexander Pautsch
  7. Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA

    • Pil Seok Chae
    •  & Samuel H. Gellman
  8. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA

    • Brian T. DeVree
    •  & Roger K. Sunahara

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Contributions

S.G.F.R. screened and characterized high affinity agonists, identified and determined dissociation rate of BI-167107, screened, identified and characterized MNG-3, performed selection and characterization of nanobodies, purified and crystallized the receptor with Nb80 in LCP, optimized crystallization conditions, grew crystals for data collection, reconstituted receptor in HDL particles and determined the effect of Nb80 and Gs on receptor conformation and ligand binding affinities, assisted with data collection and preparing the manuscript. H.-J.C. processed diffraction data, solved and refined the structure, and assisted with preparing the manuscript. J.J.F. expressed, purified, selected and characterized nanobodies, purified and crystallized receptor with nanobodies in bicelles, assisted with growing crystals in LCP, and assisted with data collection. E.P. performed immunization, cloned and expressed nanobodies, and performed the initial selections. J.S. supervised nanobody production. P.S.K. and S.H.G. provided MNG-3 detergent for stabilization of purified β2AR. B.T.D. and R.K.S. provided ApoA1 and Gs protein, and reconstituted β2AR in HDL particles with Gs. D.M.R. characterized the usefulness of MNG-3 for crystallization in LCP and assisted with manuscript preparation. F.S.T. expressed β2AR in insect cells and with T.S.K performed the initial stage of β2AR purification. A.P., A.S. assisted in selection of the high-affinity agonist BI-167107. I.K. synthesized BI-167,107. P.C. characterized the functional properties of BI-167,107 in CHO cells. W.I.W. oversaw data processing, structure determination and refinement, and assisted with writing the manuscript. B.K.K. was responsible for the overall project strategy and management, prepared β2AR in lipid vesicles for immunization, harvested and collected data on crystals, and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to William I. Weis or Brian K. Kobilka.

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DOI

https://doi.org/10.1038/nature09648

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