Fine-Tuning of the Neuropeptide Y1 G Protein-Coupled Receptor by the Tryptophan6.48 "Toggle Switch".

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 W6.48 of transmembrane helix 6 can play a role in GPCR activation, where W6.48 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 W6.48 in the neuropeptide Y1 receptor (Y1R). Via NMR experiment and molecular dynamics simulations, we find that on the one hand, W6.48 exhibits multiple rotameric conformations, where simulations indicate that these are coupled to backbone structure. On the other hand, mutation of W6.48 to alanine does not prevent G-protein signaling, and agonist vs antagonist bound Y1R exhibits the same W6.48 rotameric state, calling into question whether its core function is to regulate Y1R activation. Further investigation indicates that the W6.48 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 W6.48 in regulating Y1R binding, in which rotameric changes for W6.48 influence ligand binding by favoring backbone structures in apo Y1R that are similar to those observed for the active protein. Mutation of W6.48 then does not eliminate these structures but reduces their prevalence. Therefore, W6.48 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.