Conformational Changes in GPCR Surface and Core Probed by [13C]-Methyl NMR Spectroscopy

Abstract

Recent crystal structures reveal the inactive states of non-rhodopsin G-protein coupled receptors (GPCRs) in beautiful detail. Solution NMR spectroscopy is ideally suited to contribute dynamic information regarding GPCR activation. However, these eukaryotically-expressed membrane proteins remain challenging NMR targets. We apply selective labeling with [13C]methyl probes and two-dimensional NMR to analyze ligand-induced conformational changes in beta2-adrenergic receptor (b2AR).Lysine side chains were labeled with [13C]dimethyl probes to explore conformational changes in the b2AR extracellular surface. Lys305 forms a salt bridge connecting the extracellular end of transmembrane (TM) helix 7 with extracellular loop 2. The Lys305 NMR resonances are sensitive to conformational changes in the receptor extracellular surface. Using NMR, we observe disruption of the Lys305 salt bridge upon receptor activation by agonist. Computational modeling suggests that a lateral displacement of TM7 occurs in concert with an inward motion at the extracellular end of TM6 (thus extending the “global toggle switch” model of Schwartz (2006) Annu. Rev. Pharmacol. Toxicol.) Different conformational changes occur upon inverse agonist binding. Molecular dynamics simulations suggest that a conserved phenylalanine (Phe193) in the orthosteric ligand binding site is key for inverse agonism. Taken as a whole, these results demonstrate conformational coupling between the GPCR extracellular surface and orthosteric ligand binding site within the transmembrane domains (Ahuja (2009) Nat. Struct. Mol. Biol.) This provides rationale for developing allosteric pharmaceuticals targeting the GPCR extracellular surface.Conformational changes within the b2AR transmembrane core are also observed by NMR using selective epsilon-[13CH3] labeling of methionines. While assignments are pending, clear conformational changes are seen with activation or inverse agonist binding. [13C]methyl NMR spectroscopy, in combination with crystal structures and molecular dynamics simulation, provides a dynamic view of the conformational changes intrinsic to GPCR function.