Actin-Binding Compounds that Affect the Weak-To-Strong Actin-Myosin Interaction

Abstract

We measured the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-iodoacetamide-actin (PIA). These compounds, previously identified from a small-molecule screen involving lifetime-detected FRET from fluorescently-labeled actin to a myosin-derived actin-binding peptide, perturb actin-activated myosin ATPase and microsecond structural dynamics of actin (Guhathakurta et. al. 2018, JBC 293:12288). In the present study, several of these compounds were evaluated by measuring their effects on PIA fluorescence, which is quenched specifically by the strong binding (post-powerstroke state) of myosin to actin. In the absence of S1, several compounds reduced the steady-state fluorescence of the compound-PIA complex, indicating an increase in the compound-induced structural change and/or the compound's affinity for actin. Excess MgATP partially restored the fluorescence. In the absence of ATP and compound, the addition of S1 to PIA decreased fluorescence substantially, indicating strong binding, while the addition of MgATP reversed this effect. However, in several cases, the compound partially prevented the restoration of S1-PIA fluorescence by MgATP, suggesting that a fraction of actin either retained strongly bound S1 or maintained the compound-induced structural change as in the absence of S1. In some cases, higher [ATP] was required to increase the PIA fluorescence, suggesting that the compound decreased ATP affinity. Transient kinetics measurements are ongoing to determine the effect of compounds on the ATP-induced release of S1 from the compound-PIA complex, which will provide insight on how actin-myosin interactions are perturbed by these compounds, some of which are currently used for therapeutic purposes. This work was supported by NIH grants to DDT (R01AR032961, R01HL129814, and R37AG026160).