Structure and function of an irreversible agonist-β2 adrenoceptor complex

Top Cited Papers
Open Access
Publisher Website
Google Scholar
Abstract
Two papers by Brian Kobilka and colleagues describe the X-ray crystal structure of the human β2 adrenergic receptor (β2AR) bound to various agonists. β2AR is a member of the G protein coupled receptor (GPCR) family of membrane-spanning receptors that sense molecules outside the cell and activate internal signalling pathways. With a ubiquitous role in human physiology, GPCRs are prime targets for drug discovery. A third paper by Christopher Tate and his team describes crystal structures of a similar GPCR, the turkey β1-adrenergic receptor (β1AR), bound to full and partial agonists. Together, these new structures reveal the subtle structural changes that accompany agonist binding, showing how binding events inside and outside the cell membrane stabilize the receptor's active state. Agonist binding to β1AR is shown to induce a contraction of the catecholamine-binding pocket relative to the antagonist-bound receptor, and molecular-dynamics simulations of the β2AR agonist complex suggest that the agonist-bound active state spontaneously relaxes to an inactive-like state in the absence of a G protein. The X-ray crystal structure of the human β2 adrenergic receptor, a G-protein-coupled receptor (GPCR), covalently bound to a small-molecule agonist is solved. Comparison of this structure with structures of this GPCR in an inactive state and in an antibody-stabilized active state reveals how binding events at both the extracellular and intracellular surfaces stabilize the active conformation of the receptor. Molecular dynamics simulations suggest that the agonist-bound active state spontaneously relaxes to an inactive-like state in the absence of a G protein. 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.