Activation mechanism of the β2-adrenergic receptor

Abstract

A third of marketed drugs act by binding to a G-protein-coupled receptor (GPCR) and either triggering or preventing receptor activation. Although recent crystal structures have provided snapshots of both active and inactive functional states of GPCRs, these structures do not reveal the mechanism by which GPCRs transition between these states. Here we propose an activation mechanism for the β(2)-adrenergic receptor, a prototypical GPCR, based on atomic-level simulations in which an agonist-bound receptor transitions spontaneously from the active to the inactive crystallographically observed conformation. A loosely coupled allosteric network, comprising three regions that can each switch individually between multiple distinct conformations, links small perturbations at the extracellular drug-binding site to large conformational changes at the intracellular G-protein-binding site. Our simulations also exhibit an intermediate that may represent a receptor conformation to which a G protein binds during activation, and suggest that the first structural changes during receptor activation often take place on the intracellular side of the receptor, far from the drug-binding site. By capturing this fundamental signaling process in atomic detail, our results may provide a foundation for the design of drugs that control receptor signaling more precisely by stabilizing specific receptor conformations.

Fig. 1.

The β₂AR G-protein-binding site adopts three major conformations: active, inactive, and a previously unobserved intermediate. (A) The distance between Arg131^3.50 and Leu272^6.34 C_α atoms (a measure of helix 6 displacement) and the rmsd of Asn322^7.49–Cys327^7.54 backbone atoms [a measure of helix 7 conformation; relative to the inactive structure (PDB entry 2RH1)] are plotted for a simulation in which an agonist-bound receptor, initially in the conformation of the active structure (PDB entry 3P0G) spontaneously transitioned to an inactive conformation. Circles represent simulation snapshots sampled every 6 ns. Diamonds correspond to the active (3P0G) and inactive (2RH1) crystal structures, and to the average value of these coordinates in a simulation of the inactive structure with the cocrystallized inverse agonist bound (2RH1-sim; Table S1, condition R). (B) Representative structures of the three major conformational clusters, each superimposed on the inactive structure (light blue). Data are from simulation 11 (Table S2).

Fig. 2.

The allosteric network underlying β₂AR activation involves three regions that each switch individually between discrete conformations. (A) Active structure of β₂AR, with three key regions highlighted. (B) Representative structures of the dominant conformations for each region in simulation. (C) Quantities indicative of each region’s conformation: the vectorial displacement of the Ser207^5.46 C_α atom away from helix 7 (see SI Text) and the distance between the Ser207^5.46 side-chain oxygen and agonist p-phenol oxygen atoms (ligand-binding site); the rmsd from the active and inactive structures of the nonsymmetric, non-hydrogen atoms in Ile121^3.40 and Phe282^6.44 (connector); and the two quantities plotted in Fig. 1A (G-protein-binding site). The time series are smoothed with a 9.9-ns running average. These quantities and related ones are used to classify the conformation of each region (see SI Text). The shaded bars below the plots indicate which conformation is adopted by each region as a function of time (active/inactive for the ligand-binding site and connector; active/intermediate/inactive for the G-protein-binding site), with color coding matching that of B. Data are from simulation 11 (Table S2).

Fig. 3.

The ligand- and G-protein-binding sites are loosely coupled, but an inactive G-protein-binding site restricts the connector to its inactive conformation. Each set of three horizontal bars indicates which conformation is adopted by the ligand-binding site, the connector, and the G-protein-binding site during that simulation. The upper inset shows which conformations are adopted in each major receptor state in our agonist-bound simulations; large text indicates a predominant conformation. Simulation numbers match those in Table S2. The final simulation was initiated from the inactive structure, with the inverse agonist carazolol bound (Table S1, condition R).

Fig. 4.

A typical β₂AR activation pathway as inferred from our simulations. In A, an agonist has bound to an inactive receptor. (B) The activation process begins on the receptor’s intracellular side with an outward motion of helix 6, past Tyr219^5.58, bringing the receptor to an intermediate state. Here, the connector and the ligand-binding site are in equilibrium between inactive and active conformations; the bound agonist stabilizes the active conformations. (C) A G protein may bind to the intermediate state, favoring the final step on the activation pathway: conformational change in the NPxxY motif (helix 7) that positions the side chain of Tyr326^7.53 close to that of Tyr219^5.58. Other activation pathways are also possible.