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Crystal structure of the β2 adrenergic receptor–Gs protein complex

Nature volume 477, pages 549–555 (2011)Cite this article

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Abstract

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

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Figure 1: G protein cycle for the β 2 AR–Gs complex.
Figure 2: Overall structure of the β 2 AR–Gs complex.
Figure 3: Comparison of active and inactive β 2 AR structures.
Figure 4: Receptor-G protein interactions.
Figure 5: Conformational changes in Gαs.
Figure 6: Possible sequence of β 2 AR–Gs complex formation.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the β2AR–Gs complex are deposited in the Protein Data Bank (accession code 3SN6).

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Acknowledgements

We acknowledge support from National Institutes of Health Grants NS028471 (B.K.K.) and GM083118 (B.K.K. and R.K.S.), GM56169 (W.I.W.), P01 GM75913 (S.H.G), T32-GM008270 and P60DK-20572 (R.K.S.), GM75915, P50GM073210 and U54GM094599 (M.C.), Science Foundation Ireland (07/IN.1/B1836) and FP7 COST Action CM0902 (M.C.),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.), The Danish Council for Independent Research, Medical Sciences (J.M.M.). We thank D. Grayson and A. Coughlan for help with lipid synthesis.

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  1. Søren G. F. Rasmussen and Brian T. DeVree: These authors contributed equally to this work.

Authors and Affiliations

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

    Søren G. F. Rasmussen, Yaozhong Zou, Andrew C. Kruse, Ka Young Chung, Tong Sun Kobilka, Foon Sun Thian, Jesper M. Mathiesen, William I. Weis & Brian K. Kobilka

  2. Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark

    Søren G. F. Rasmussen

  3. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, 48109, Michigan, USA

    Brian T. DeVree, Diane Calinski & Roger K. Sunahara

  4. Department of Chemistry, University of Wisconsin, Madison, 53706, Wisconsin, USA

    Pil Seok Chae & Samuel H. Gellman

  5. Department of Molecular and Cellular Interactions, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel, B-1050 Brussel, Belgium

    Els Pardon & Jan Steyaert

  6. Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium

    Els Pardon & Jan Steyaert

  7. Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry & Immunology, Trinity College, Dublin 2, Ireland

    Syed T. A. Shah, Joseph A. Lyons & Martin Caffrey

  8. Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Georgios Skiniotis

  9. Department of Structural Biology, Stanford University School of Medicine, Stanford, 94305, California, USA

    William I. Weis

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  1. Søren G. F. Rasmussen
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Contributions

S.G.F.R. performed the final stages of β2AR purification; assisted with β2AR and Gs protein virus production and expression in insect cell cultures; worked out conditions to form and stabilize the β2AR–Gs complex following screening, identification and characterization of the BI-167107 agonist and MNG-3 detergent; developed the β2AR–Gs complex purification strategy with B.K.K. and characterized the stability of the complex under a variety of conditions; purified and analysed all preparations of the β2AR–Gs complex used for crystallography, DXMS and electron microscopy studies, immunization, and nanobody selection; expressed and purified nanobodies and characterized β2AR–Gs–Nb binding by size exclusion chromatography; set up crystallization trials in detergent solution, bicelles and lipidic cubic phase; crystallized the T4L–β2AR–Gs, T4L–β2AR–Gs–Nb37 and T4L–β2AR–Gs–Nb35 complexes; optimized crystallization conditions and grew crystals for data collection; assisted with data collection and manuscript preparation. B.T.D. managed Gs heterotrimer subunit virus production and titration; expressed and purified Gs protein; with R.K.S. he identified the use of apyrase in forming the β2AR–Gs complex and foscarnet/pyrophosphate during crystallogenesis; reconstituted the β2AR–Gs complex and receptor alone in high density lipoprotein particles which were used for the initial nanobody selection. He assisted with data collection. Y.Z. designed, generated and optimized the T4L–β2AR fusion protein, characterized its expression and functional properties, and prepared baculovirus for large scale expression. A.C.K. collected crystals, collected and processed diffraction data, solved and refined the structure, and assisted with manuscript preparation. K.Y.C. developed the cross-linking conditions for the purified β2AR–Gs complex used for immunization of llamas. E.P. performed llama immunization, cloned and expressed nanobodies and performed initial selections. J.S. supervised nanobody production. D.C. assisted with Gs heterotrimer expression and purification. J.M.M. generated the β2AR–Gsα peptide fusion construct, expressed it in insect cell membranes and performed competition binding experiments. F.S.T. expressed β2AR in insect cell cultures and with T.S.K. performed the initial stage of β2AR purification. S.T.A.S., J.A.L. and M.C. provided the 7.7 MAG lipid and helpful suggestions for lipidic mesophase crystallization using this lipid. P.S.C. and S.H.G. provided MNG-3 detergent for stabilization of the β2AR–Gs complex. G.S. provided the essential feedback from the electron microscopy studies that helped guide the crystallization effort. W.I.W. oversaw data processing, structure determination and refinement. R.K.S. supervised Gs protein production, provided valuable ideas and insights into Gs structure and function, and assisted with data collection and manuscript preparation. B.K.K. was responsible for overall project strategy and management, harvested crystals and assisted with collection of diffraction data, and wrote the manuscript.

Corresponding authors

Correspondence to Roger K. Sunahara or Brian K. Kobilka.

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Rasmussen, S., DeVree, B., Zou, Y. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011). https://doi.org/10.1038/nature10361

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