Structure and function of an irreversible agonist-β(2) 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 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.

Figure 1. Design and function of a covalent agonist

a, Structure of the carazolol-bound β₂AR, receptor in gray cartoon and ligand in yellow sticks, showing distance between isopropyl group and His93^2.64 imidazole. b, Structures of carazolol and the related BABC ligand, covalent ligand FAUC50 and noncovalent analog FAUC72. c, G protein activation assay demonstrating that covalently bound FAUC50 activates the β₂AR.

Figure 2. Comparison of agonist bound β₂AR structures

Comparison of the covalent FAUC50-bound β₂AR^H93CT4L (left panels, blue cartoon, ligand carbons in purple), BI-167107-bound β₂AR-T4L/nanobody (β₂AR-Nb80) complex (middle panels, orange cartoon, ligand carbons in green), and carazolol-bound β₂AR-T4L (right panels, cyan cartoon, ligand carbons in yellow). a, Hormone binding site with interactions between ligands and receptors. TMs 6 and 7 with residues Phe289, Asn293, and Try308 are omitted for clarity. b, Comparison of the cytoplasmic surfaces showing differences in TMs 5 and 6. Superimposed β₂AR-Nb80 complex is also shown in left panel as a transparent cartoon, and arrows indicate rigid body movements. c, Conformational switch region with residues Ile121^3.40, Pro211^5.50, and Phe282^6.44 from TMs 3, 5, and 6 (other TMs transparent). Sidechains are shown in van der Waals sphere representation. A dashed line is shown at an equivalent position in each receptor.

Figure 3. Molecular dynamics simulations

a, An unbiased simulation initiated from the nanobody complex (β₂AR-Nb80) structure, with the agonist BI-167107 bound but the nanobody removed (magenta), and a carazolol-bound simulation initiated from the inactive structure (β₂AR-Cz) (blue). (top) Distance between C_α atoms of Arg131^3.50 and Leu272^6.34, and (bottom) RMSD of non-symmetric non-hydrogen atoms in residues Ile121^3.40 and Phe282^6.44. Dashed lines indicate corresponding quantities from crystal structures. b, Cytoplasmic view of the simulated agonist-bound receptor after 30 μs, compared to the β₂AR-Nb80 (left) and β₂AR-FAUC50 (right) structures. The conformations of intracellular loop 2, Tyr219^5.58, and Glu268^6.30 shown for the agonist-bound simulation differ from β₂AR-FAUC50, but have been observed in inactiveβ₂AR simulations and in other inactive-state GPCR structures. c, Proposed energy landscape model, in which both an agonist and a cytoplasmic binding partner are required to stabilize the fully active receptor conformation [R*] over intermediate [R′ and R″] and inactive [R] states.