Probing Allosteric Regulation Mechanism of W7.35 on Agonist-Induced Activity for µOR by Mutation Simulation.

ABSTRACT

The residue located at 15 positions before the most conserved residue in TM7 (7.35 of Ballesteros-Weinstein number) plays important roles in ligand binding and the receptor activity for class A GPCRs. Nevertheless, its regulation mechanism has not been clearly clarified in experiments, and some controversies also exist for its impact on µ-opioid receptors (µOR) bound by agonists. Thus, we chose the µ-opioid receptor (µOR) of class A GPCRs as a representative and utilized a microsecond accelerated molecular dynamics simulation (aMD) coupled with a protein structure network (PSN) to explore the effect of W3187.35 on its functional activity induced by the agonist endomorphin2 mainly by a comparison of the wild system and its W7.35A mutant. When endomorphin2 binds to the wild-type µOR, TM6 in µOR moves outward to form an open intracellular conformation that is beneficial to accommodating the ß-arrestin transducer, rather than the G-protein transducer due to the clash with the α5 helix of G-protein, thus acting as a ß-arrestin biased agonist. However, the W318A mutation induces the intracellular part of µOR to form a closed state, which disfavors coupling with either G-protein or ß-arrestin. The allosteric pathway analysis further reveals that the binding of endomorphin2 to the wild-type µOR transmits more activation signals to the ß-arrestin binding site while the W318A mutation induces more structural signals to transmit to common binding residues of the G protein and ß-arrestin. More interestingly, the residue at the 7.35 position regulates the shortest allosteric pathway in indirect ways by influencing the interactions between other ligand-binding residues and endomorphin2. W2936.48 and F2896.44 are important for regulating the different activities of µOR induced either by the agonist or by the mutation. Y3367.53, F3438.50, and D3408.47 play crucial roles in activating the ß-arrestin biased signal induced by the agonist endomorphin2, while L1583.43 and V2866.41 devote important contributions to the change in the activity of endomorphin2 from the ß-arrestin biased agonist to the antagonist upon the W318A mutation.