Allosteric inhibition of myosin by phenamacril: a synergistic mechanism revealed by computational and experimental approaches

Abstract

BackgroundMyosin plays a crucial role in cellular processes, while its dysfunction can lead to organismal malfunction. Phenamacril (PHA), a highly species‐specific and non‐competitive inhibitor of myosin I (FgMyoI) from Fusarium graminearum, has been identified as an effective fungicide for controlling plant diseases caused by partial Fusarium pathogens, such as wheat scab and rice bakanae. However, the molecular basis of its action is still unclear.ResultsThis study employed multiple computational approaches first to elucidate the allosteric inhibition mechanism of FgMyoI by PHA at the atomistic level. The results indicated the increase of ATP binding affinity upon PHA binding, which might impede the release of hydrolysis products. Furthermore, simulations revealed a broadened outer cleft and a significantly more flexible interface for actin binding, accompanied by a decrease in signaling transduction from the catalytic center to the actin‐binding interface. These various effects might work together to disrupt the actomyosin cycle and hinder the ability of motor to generate force. Our experimental results further confirmed that PHA reduces the enzymatic activity of myosin and its binding with actin.ConclusionTherefore, our findings demonstrated that PHA might suppress the function of myosin through a synergistic mechanism, providing new insights into myosin allostery and offering new avenues for drug/fungicide discovery targeting myosin.This article is protected by copyright. All rights reserved.

Characterizing the Molecular Mechanism of the Lethal C423D Mutation in FgMyoI: A Molecular Perspective

“…We compared the cleft distance between the two interface regions, and as shown in Figure , same as the allosteric inhibitor phenamacril, the mutation in this work also induced a more flexible and widening interfacial cleft loops, which has been confirmed to be disfavor with the proper anchoring of FgMyoI to the F-actin, coinciding with the inhibition of myosin-actin PPIs as observed above (Figure c,d). These mutation-induced conformational changes in myosin resemble those upon binding of allosteric inhibitor phenamacril but are more pronounced in strength, in agreement with the more severe damage caused by C423D mutation on the growth of F.…”

“…Furthermore, the C423D mutation is precisely allosterically located within the binding pocket of the allosteric inhibitor phenamacril of FgMyoI near the SW2 motif. We thus speculate that C423D might hinder the function of FgMyoI through a mechanism similar to phenamacril as revealed in our companion study . Extensive molecular dynamics (GaMD) simulations and experiments have been conducted to confirm this further.…”

“…As previously discussed, the closure of SW2 is the hallmark of the starting of myosin recovery stroke, which is characterized by the formation of the critical Pi-G410 hydrogen bond and R183-E412 salt bridge. , From the free-energy landscape of the two critical distances (Figure d–f), an opened or partially opened SW2 could be observed with a completely disrupted G410-Pi hydrogen-bond interaction and the larger R183-E412 salt-bridge distance. This is in contrast to the closure of SW2 induced by the allosteric inhibitor phenamacril . Upon inhibitor binding, the closure of SW2 allows the recovery stroke of myosin to be initiated, but the subsequent release of hydrolysis products will be inhibited.…”

“…For further confirmation, we expressed and purified the wild-type (WT) and mutated (Mut) FgMyoI. As shown from the ATPase assays (Figure b), FgMyoI exhibited strongly reduced ATPase activity upon the C423D mutation and was even more impaired than phenamacril did . The ability of FgMyoI and its mutant to interact with actin was probed by using microscale thermophoresis (MST).…”

“…Orthosteric ligands ATP and ADP-Pi were modified from the phosphothiophosphoric acid-adenylate ester (ATP analogue) of the crystal structure by Schrödinger Maestro, and residue mutation was conducted by PyMOL, with residues 40–715 of the protein considered in the simulation. Same as in our previous study, all systems were solvated with TIP4P-EW water, and sodium ions were used to neutralize the system charge. The receptor and nucleotides ATP/ADP were described by ff14SB and amber ATP/GDP force fields, respectively, and a 12–6–4 LJ-type nonbonded model , was applied for metal ion Mg 2+ .…”