Structure and function of an irreversible agonist-β2 adrenoceptor complex

Abstract

G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs1, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human β2 adrenergic receptor (β2AR) as a guide, we designed a β2AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β2AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β2AR–T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method2, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper3) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 μs) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

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Figure 1: Design and function of a covalent agonist.
Figure 2: Comparison of agonist and inverse agonist bound β 2 AR structures.
Figure 3: Molecular dynamics simulations.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for β2AR–FAUC50 are deposited in the Protein Data Bank (accession code 3PDS).

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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), GM75915 and P50GM073210 (M.C), 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.),Science Foundation Ireland (07/IN.1/B1836) and FP7 COST Action CM0902 (M.C.), the Bavaria California Technology Center (P.G.), and the University of Michigan Biomedical Sciences Scholars Program (R.K.S). We thank Stefan Löber, Harald Hübner, Albert Pan and Paul Maragakis for discussion and suggestions. We thank Foon Sun Thian for help with insect cell expression.

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D.M.R. designed project and agonists, did binding assays to characterize agonists, developed purification, optimized crystallization conditions, optimized construct, purified protein, grew and harvested crystals, collected data, solved structure, refined structure, wrote manuscript. C.Z. performed G protein activation assays for covalent agonist, prepared recombinant baculovirus, performed large-scale expression of recombinant β2AR in insect cells, purified protein, grew crystals. J.L. helped to optimize crystallization conditions, grew and harvested crystals, collected data. D.A. was involved in LCP optimization, harvested crystals, collected data. R.H. synthesized agonists. S.G.F.R. identified the use of MNG3 detergent for β2AR stabilization and assisted with manuscript preparation. H.J.C. assisted with data processing and refinement. R.K.S. and B.D. provided ApoA1 and Gs protein for functional characterization of the covalent ligand. P.S.C. and S.G. provided MNG3 detergent for stabilization of purified β2AR. D.H.A. and R.O.D. performed and analyzed MD simulations; R.O.D. and D.E.S. oversaw MD simulations and analysis. W.I.W. assisted with data processing and refinement and with manuscript preparation. M.C. helped to guide the LCP crystallization efforts at Stanford and in Ireland, and oversaw automated lipidic cubic phase crystallography screens. P.G. designed the strategy for the synthesis of the covalent agonist. B.K. was responsible for the overall project strategy and management, oversaw manuscript preparation, and assisted with synchrotron data collection.

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Correspondence to Martin Caffrey or Peter Gmeiner or Brian K. Kobilka.

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The authors declare no competing financial interests.

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Rosenbaum, D., Zhang, C., Lyons, J. et al. Structure and function of an irreversible agonist-β2 adrenoceptor complex. Nature 469, 236–240 (2011). https://doi.org/10.1038/nature09665

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