D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

Abstract

To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. 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. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. 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 actomyosin-ADP⅐P i state of both myosins. Subdomain 2 of actin, which contains the 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 (1, 2). 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 (3, 4), whereas the proteolytic digestion of the D-loop inhibits actin-activated ATPase of myosin II-subfragment 1 (S1) 3 and decreases the velocity of actin filaments on myosin II-HMM in an in vitro motility assay (5, 6).However, although as many as 24 classes of myosin have already been found (7), the contribution of the D-loop to actinmyosin interaction has so far been studied only for myosin II. Although all myosins carry out their motor functions by interacting with actin filaments, each class has a different role in vivo. For example, myosin V is an intracellular transporter, which is different from the role of myosin II, engaged mainly in muscle contraction or the formation of contractile ring. The rate-limiting step in the ATPase cycle in the presence of actin is the phosphate release in the case of myosin II but ADP release in myosin V, which allows this motor to spend most of its ATPase cycle (Ͼ90%) strongly bound to actin. Furthermore, similarly to kinesin (8), two heads of myosin V work cooperatively, coordinating their biochemical cycles via internal load (9 -11). These characteristics enable myosin V to move processively on actin filaments (12), and recent studies indicate that, similarly to myosin II, the attachment of myosin V also induces conformational changes in the D-loop region of actin (13).Here, to determine whether the D-loop contributes to the interact...

G146V Mutation at the Hinge Region of Actin Reveals a Myosin Class-specific Requirement of Actin Conformations for Motility

“…One is that because the rate-limiting step in the myosin V ATPase cycle is ADP release (40), the slower transition from weak to strong binding may not be deleterious for this myosin. Consistent with this possibility, Kubota et al (41) reported that the M47A/E360H mutation of Dictyostelium actin slows down the transition of the leading head from the weak to the strong binding state, but it does not reduce gliding velocity. The second possibility is that the conformational states of the actin subunits required for myosin V motility differ from those required for myosin II motility.…”

Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

“…It is noteworthy that Rng2CHD did not inhibit actin filament movement on myosin V HMM, which is well established to move actin filaments processively with a lever arm swing (Forkey et al, 2003, Kodera et al, 2010. In line with this finding, two actin mutations, M47A (Kubota et al, 2009) and G146V (Noguchi et al, 2012), both of which likely perturb conformational changes of actin filaments, inhibit actin movements on myosin II, but not on myosin V. We suggest that the movement by myosin II, which is a fast and non-processive motor, requires some unidentified structural property of actin filaments which is dispensable for the slow and processive myosin V involving the lever arm swing. Further studies are needed to understand the mechanism by which actin filaments structurally altered by Rng2CHD fails to move on myosin II.…”

A novel interplay between oncogenic PFTK1 protein kinase and tumor suppressor TAGLN2 in the control of liver cancer cell motility

“…In fact, tyrosine phosphorylation of actin can protect its D-loop structure from protease cleavage, resultant in a more stable conformation of the actin D-loop than the unphosphorylated form (Baek et al, 2008). Conformational stability of the D-loop holds much importance in conferring cell motile properties, which if unstable can result in a weakened contractile force and much reduced gliding velocity (Kubota et al, 2009). Our finding of reduced ACTB tyrosine phosphorylation in the PFTK1-suppressed cells would seem to be in line with the development of a less stable actin structure, which could have contributed to the repression on HCC cell migration.…”