Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin–actin interaction

Osha Roopnarine,David D Thomas

doi:10.1016/j.jbc.2021.100471

Osha Roopnarine, David D Thomas

Open Access

https://doi.org/10.1016/j.jbc.2021.100471

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Abstract

Actin–myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin–myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin–myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

Highlights

Actin is abundant in eukaryotic cells and is involved in many important cellular processes such as movement, cell division, maintenance of cellular shape, transport of vesicles, phagocytosis, and contractility [1]
Starting from compounds previously identified from highthroughput screening, we have shown that pyrene-labeled actin is sensitive to conformational changes that these compounds induce in F-actin and that these conformational changes affect the strong-binding state of myosin to actin
Many of the compounds prevent full restoration of fluorescence from the actin-subfragment 1 (S1) complex in ATP, implying that they decrease the fraction of nucleotide-bound myosin or that the actin-attached states are more populated in the presence of ATP

Summary

Introduction

Actin is abundant in eukaryotic cells and is involved in many important cellular processes such as movement, cell division, maintenance of cellular shape, transport of vesicles, phagocytosis, and contractility [1]. Actin (A) and myosin (M) bind strongly (S) in a stereospecific manner in the absence of ATP to form the actin–myosin complex (A.M), in which the myosin head (catalytic domain, CD, and light-chain domain, LCD, called lever arm) is straight (Fig. 1). Cardiac actin mutations that appear in the myosin or tropomyosin-binding regions cause either hypertrophic (HCM) or dilated (DCM) cardiomyopathies [11], increasing the motivation to discover small molecules that target actin for therapeutic treatment of actinrelated diseases [12, 13]. Small molecules have recently been investigated as potential therapeutic agents for myosin mutations that cause inherited heart disease, with particular focus on hypertrophic cardiomyopathy (HCM). The results of that study were mixed, suggesting that for those troponinbased HCM mutations, Mava would not be a suitable therapeutic candidate

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