Regulation of Myosin Motility by D-Loop of Actin

Abstract

Subdomain 2 of actin, which contains the DNase I binding loop (D-loop, residues 38-52), slightly changes its conformation during actin polymerization and interacts with the C-terminus of the adjacent subunit in actin filament. This region is suggested to be important for actin-myosin interaction: it was found that binding of myosin II induces conformational changes in subdomain 2 and the proteolytic digestion of the D-loop disturbs the motor function of myosin II. However, although as many as 24 classes of myosin have already been found and their in vivo roles are completely different, the contribution of the D-loop to actin-myosin interaction has so far been studied only for myosin II.In this study, to determine whether the D-loop contributes to the interaction with myosin V and if so, in what way it affects its motor function, we prepared actins modified in the D-loop and analyzed the effects of modifications on the motile properties of myosins II and V. We found that the D-loop modifications, namely, the proteolytic digestion with subtilisin and the M47A point mutation, significantly decreased the gliding velocity on myosin II-HMM in an in vitro motility assay, due to a weaker generated force. On the other hand, single molecules of myosin V walked with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications.These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the A.M.ADP.Pi state of both myosins. Our findings are important to understand the principles how an actin molecule may regulate diverse in vivo functions of various myosin isoforms.