High Pressure Shifts the β1-Adrenergic Receptor to the Active Conformation in the Absence of G Protein.

G protein-coupled receptors (GPCRs) are versatile chemical sensors, which transmit the signal of an extracellular binding event across the plasma membrane to the intracellular side. This function is achieved via the modulation of highly dynamical equilibria of various conformational receptor states. Here we have probed the effect of pressure on the conformational equilibria of a functional thermostabilized β1-adrenergic GPCR (β1AR) by solution NMR. High pressure induces a large shift in the conformational equilibrium (midpoint ∼600 bar) from the preactive conformation of agonist-bound β1AR to the fully active conformation, which under normal pressure is only populated when a G protein or a G protein-mimicking nanobody (Nb) binds to the intracellular side of the β1AR·agonist complex. No such large effects are observed for an antagonist-bound β1AR or the ternary β1AR·agonist·Nb80 complex. The detected structural changes of agonist-bound β1AR around the orthosteric ligand binding pocket indicate that the fully active receptor occupies an ∼100 Å3 smaller volume than that of its preactive form. Most likely, this volume reduction is caused by the compression of empty (nonhydrated) cavities in the ligand binding pocket and the center of the receptor, which increases the ligand receptor interactions and explains the ∼100-fold affinity increase of agonists in the presence of G protein. The finding that isotropic pressure induces a directed motion from the preactive to the fully active GPCR conformation provides evidence of the high mechanical robustness of this important functional switch.