Key result
The W318A mutation in the μ-opioid receptor induces a closed intracellular state that disfavors coupling with G-protein or β-arrestin, altering the allosteric signaling pathway of endomorphin2.
Why the study?
The regulation mechanism of residue 7.35 in class A GPCRs has not been clearly clarified experimentally, and controversies exist regarding its impact on agonist-bound mu-opioid receptors.
Molecular dynamics simulations reveal that the W318A mutation in the μ-opioid receptor alters its conformation to a closed state, preventing G-protein and β-arrestin coupling and converting endomorphin2 into an antagonist.
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 W318 7.35 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 β-arrestin transducer, rather than the G-protein transducer due to the clash with the α5 helix of G-protein, thus acting as a β-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 β-arrestin. The allosteric pathway analysis further reveals that the binding of endomorphin2 to the wild-type μOR transmits more activation signals to the β-arrestin binding site while the W318A mutation induces more structural signals to transmit to common binding residues of the G protein and β-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. W293 6.48 and F289 6.44 are important for regulating the different activities of μOR induced either by the agonist or by the mutation. Y336 7.53, F343 8.50, and D340 8.47 play crucial roles in activating the β-arrestin biased signal induced by the agonist endomorphin2, while L158 3.43 and V286 6.41 devote important contributions to the change in the activity of endomorphin2 from the β-arrestin biased agonist to the antagonist upon the W318A mutation.
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Zhang et al. (2021) studied this question. W318A mutation and endomorphin2 binding vs. Wild-type μ-opioid receptor was evaluated on Receptor conformation and allosteric pathway activation. The W318A mutation in the μ-opioid receptor induces a closed intracellular state that disfavors coupling with G-protein or β-arrestin, altering the allosteric signaling pathway of endomorphin2.
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