Abstract
G protein-coupled receptors (GPCRs) transduce extracellular signals into the cell through binding and activation of intracellular effector proteins. Highly conserved residues such as tryptophan W(6.48) of transmembrane helix 6 can play a role in GPCR activation, where W(6.48) acts as a microswitch, changing its rotameric state depending on whether the receptor is bound to an agonist or antagonist. However, its exact role is not entirely clear. Here we investigate the role of W(6.48) in the neuropeptide Y1 receptor (Y1R). Via NMR experiment and molecular dynamics simulations, we find that on the one hand, W(6.48) exhibits multiple rotameric conformations, where simulations indicate that these are coupled to backbone structure. On the other hand, mutation of W(6.48) to alanine does not prevent G-protein signaling, and agonist vs antagonist bound Y1R exhibits the same W(6.48) rotameric state, calling into question whether its core function is to regulate Y1R activation. Further investigation indicates that the W(6.48) rotameric state restricts microstates accessible by Y1R and impacts backbone dynamics. Using principal component analysis of multiple MD trajectories, we determine the structural similarity between Y1R for the various apo, NPY, NPY/Gi, and antagonist-bound conformational states. We propose a role for W(6.48) in regulating Y1R binding, in which rotameric changes for W(6.48) influence ligand binding by favoring backbone structures in apo Y1R that are similar to those observed for the active protein. Mutation of W(6.48) then does not eliminate these structures but reduces their prevalence. Therefore, W(6.48) provides "fine-tuning" of Y1R signaling; mutation of the 6.48 position leaves Y1R active but "detuned" such that signaling is still possible, but proper functioning is inhibited by a decrease in ligand binding rate.