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 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. 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 β-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 β-arrestin biased agonist to the antagonist upon the W318A mutation.

Biased Activation Mechanism Induced by GPCR Heterodimerization: Observations from μOR/δOR Dimers

“…W6.48 and F6.44 are highly conserved activation switches for the class A GPCRs, and their important roles in GPCR activation have been confirmed. ,− Besides, MD simulations indicated that W6.48 significantly affects the β-arrestin signaling of δOR . L3.43 was also reported to associate with the β-arrestin signaling of μOR by MD simulation . Y7.53 belongs to the NPxxY motif, which is conserved in the class A GPCRs and is an important region involved in the activation of class A GPCRs .…”

“…It was revealed that the residue located at position 7.35 is a vital ligand binding residue in class A GPCRs, which plays an important role in the activation of the receptors and selective recruitment of transducers. − In the opioid receptor, the difference in the residue at position 7.35 between κOR and μOR results in distinct biased signaling even binding to the same ligand . Experiments and MD simulations on the μOR monomer found that the W7.35A mutation changes endomorphin2 from a β-arrestin-biased agonist of μOR to an antagonist. Our observation from the dimer further confirms the importance of the residue in the ligand binding to the heterodimer and speculates that W7.35 is also a key residue that alters the signaling efficacy of DAMGO in the μOR/δOR heterodimers.…”

Biased Signaling in Mutated Variants ofβ2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations

“…It is well known that the perturbations, including ligand/transducer binding, 26,33,34,37,40,86,121 post-translational modifications such as phosphorylation, 44,129 and mutation, 19 trigger conformational changes leading to activation and biased signaling of GPCRs. The distinct reconfigurations of allosteric pathways, connecting the extracellular agonist and different transducer interfaces, due to mutations, are suggestive of driving specific conformations of β 2 AR that supports the observed bias in experiments.…”

“…We further note that consistent with our findings, the TM6 residues F282 6.44 , W286 6.48 , and F289 6.51 also appeared in the allosteric paths from the β-arrestin-biased agonist ethylnorepinephrine to the intracellular regions in a simulation study of β2AR. 33 It is well known that the perturbations, including ligand/transducer binding, 26,33,34,37,40,86,121 posttranslational modifications such as phosphorylation, 44,129 and mutation, 19 trigger conformational changes leading to activation and biased signaling of GPCRs. The distinct reconfigurations of allosteric pathways, connecting the extracellular agonist and different transducer interfaces, due to mutations, are suggestive of driving specific conformations of β2AR that supports the observed bias in experiments.…”

Computational Chemistry in Asia

“…Chen et al 13 used a randomly accelerated MD algorithm and funnel meta-dynamic simulations to explore the dissociation mechanisms of nonsteroidal GR ligands. Gaussian accelerated MD simulations were coupled with a dynamic network to probe the mechanisms of distinct biased activation induced by structural variations by Chen et al 14 Zhang et al 15 applied microsecond accelerated MD simulations coupled with a protein structure network to explore allosteric regulation mechanisms. Moreover, other well-established methods also play an important role in mechanism studies.…”