Structure of a nanobody-stabilized active state of the β2 adrenoceptor

Journal name:
Nature
Volume:
469,
Pages:
175–180
Date published:
DOI:
doi:10.1038/nature09648
Received
Accepted
Published online

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 11Å 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.

At a glance

Figures

  1. Effect of Nb80 on [bgr]2AR structure and function.
    Figure 1: Effect of Nb80 on β2AR structure and function.

    a, The cartoon illustrates the movement of the environmentally-sensitive bimane probe attached to Cys2656.27 in the cytoplasmic end of TM6 from a more buried, hydrophobic environment to a more polar, solvent-exposed position during receptor activation that results in a decrease in fluorescence in Fig. 1b–c and Supplementary Fig. 2c, d. b, c, Fluorescence emission spectra showing ligand-induced conformational changes of monobromobimane-labelled β2AR reconstituted into high density lipoprotein particles (mBB-β2AR/HDL) in the absence (black solid line) or presence of full agonist isoproterenol (ISO, green wide dashed line), inverse agonist ICI-118,551 (ICI, black dashed line), Gs heterotrimer (red solid line), nanobody-80 (Nb80, blue solid lines), and combinations of Gs with ISO (red wide dashed line), Nb80 with ISO (blue wide dashed line), and Nb80 with ICI (blue dashed line). d−f, Ligand binding curves for ISO competing against [3H]-dihydroalprenolol ([3H]-DHA) for d, β2AR/HDL reconstituted with Gs heterotrimer in the absence or presence GTPγS; e, β2AR/HDL in the absence and presence of Nb80; and f, β2AR–T4L/HDL in the absence and presence of Nb80. Error bars represent standard errors.

  2. Comparison of the agonist-Nb80 stabilized crystal structures of the [bgr]2AR with inverse agonist bound [bgr]2AR and opsin.
    Figure 2: Comparison of the agonist-Nb80 stabilized crystal structures of the β2AR with inverse agonist bound β2AR and opsin.

    The structure of inverse agonist carazolol-bound β2AR–T4L (β2AR–Cz) is shown in blue with the carazolol in yellow. The structure of BI-167107 agonist-bound and Nb80-stabilized β2AR–T4L (β2AR–Nb80) is shown in orange with BI-167107 in green. These two structures were aligned using the PyMOL align function. a, Side view of the β2AR–Nb80 complex with β2AR in orange and CDRs of Nb80 in light blue (CDR1) and blue (CDR3). b, Side view of the superimposed structures showing significant structural changes in the intracellular and G protein facing part of the receptors. c, Comparison of the extracellular ligand binding domains showing modest structural changes. d, Cytoplasmic view showing the ionic lock interaction between Asp3.49 and Arg3.50 of the DRY motif in TM3 is broken in the β2AR–Nb80 structure. The intracellular end of TM6 is moved outward and away from the core of the receptor. The arrow indicates an 11.4Å change in distance between the α-carbon of Glu6.30 in the structures of β2AR–Cz and β2AR–Nb80. The intracellular ends of TM3 and TM7 move towards the core by 4 and 2.5Å, respectively, while TM5 moves outward by 6Å. e, The β2AR–Nb80 structure superimposed with the structure of opsin crystallized with the C-terminal peptide of Gt (transducin)2. PyMOL (http://www.pymol.org) was used for the preparation of all structure figures.

  3. Ligand binding pocket of BI-167107 and carazolol-bound [bgr]2AR structures.
    Figure 3: Ligand binding pocket of BI-167107 and carazolol-bound β2AR structures.

    a, b, Extracellular views of the agonist BI-167107-bound (a) and carazolol-bound (b) structures, respectively. Residues within 4Å of one or both ligands are shown as sticks. In all panels, red and blue represent oxygen and nitrogen, respectively. c, d, Schematic representation of the interactions between the β2AR and the ligands BI-167107 (c) and carazolol (d). The residues shown here have at least one atom within 4Å of the ligand in the crystal structures. Mutations of amino acids in orange boxes have been shown to disrupt both antagonist and agonist binding. Mutations of amino acids in blue boxes have been shown to disrupt agonist binding. Green lines indicate potential hydrophobic interactions and orange lines indicate potential polar interactions.

  4. Rearrangement of transmembrane segment packing interactions upon agonist binding
    Figure 4: Rearrangement of transmembrane segment packing interactions upon agonist binding

    a, The BI-167107- and carazolol-bound structures are superimposed to show structural differences propagating from the ligand-binding pocket. BI-167107 and carazolol are shown in green and yellow, respectively. b, Packing interactions that stabilize the inactive state are observed between Pro211 in TM5, Ile121 in TM3, Phe282 in TM6 and Asn318 in TM7. c, The inward movement of TM5 upon agonist binding destabilizes the packing of Ile121 and Pro211, resulting in a rearrangement of interactions between Ile121 and Phe282. These changes contribute to a rotation and outward movement of TM6 and an inward movement of TM7.

Accession codes

Primary accessions

Protein Data Bank

References

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Author information

  1. These authors contributed equally to this work.

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

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

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 financial interests

The authors declare no competing financial interests.

Corresponding authors

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

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Supplementary information

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  1. Supplementary Information (3.9M)

    The file contains Supplementary Figures 1-6 with legends, Supplementary Tables 1-2 and additional references.

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