TY - JOUR

T1 - Structure and function of an irreversible agonist-β(2) adrenoceptor complex

AU - Rosenbaum, Daniel M

AU - Zhang, Cheng

AU - Lyons, Joseph A

AU - Holl, Ralph

AU - Aragao, David

AU - Arlow, Daniel H

AU - Rasmussen, Søren Gøgsig Faarup

AU - Choi, Hee-Jung

AU - Devree, Brian T

AU - Sunahara, Roger K

AU - Chae, Pil Seok

AU - Gellman, Samuel H

AU - Dror, Ron O

AU - Shaw, David E

AU - Weis, William I

AU - Caffrey, Martin

AU - Gmeiner, Peter

AU - Kobilka, Brian K

PY - 2011/1/13

Y1 - 2011/1/13

N2 - 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 GPCRs, 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 β(2) adrenergic receptor (β(2)AR) as a guide, we designed a β(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) 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.

AB - 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 GPCRs, 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 β(2) adrenergic receptor (β(2)AR) as a guide, we designed a β(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent β(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound β(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) 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.

KW - Adrenergic beta-2 Receptor Agonists

KW - Crystallization

KW - Crystallography, X-Ray

KW - Disulfides

KW - Drug Inverse Agonism

KW - Heterotrimeric GTP-Binding Proteins

KW - Humans

KW - Lipid Bilayers

KW - Models, Molecular

KW - Molecular Dynamics Simulation

KW - Procaterol

KW - Propanolamines

KW - Protein Conformation

KW - Receptors, Adrenergic, beta-2

KW - Recombinant Fusion Proteins

KW - Viral Proteins

U2 - 10.1038/nature09665

DO - 10.1038/nature09665

M3 - Journal article

C2 - 21228876

VL - 469

SP - 236

EP - 240

JO - Nature

JF - Nature

SN - 0028-0836

IS - 7329

ER -