The Substep of Myosin Va is Associated with an Increase in Stiffness of the Myosin Va Head Domain

Abstract

Vertebrate myosin Va transports cargo by processive movement that is optimized by coordination of the two heads via intramolecular strain (Veigel et al Nat Cell Biol 2002). Such coordination requires substantial stiffness of the head domains. Otherwise one head cannot “sense” changes in the other head. Substantial stiffness, however, generates substantial restoring forces impeding diffusive movement of the free head to its next binding site about 10nm beyond the throw generated by the stroke of the myosin Va. Such conflicting constraints raise the question whether stiffness of the myosin Va head domain may change during the ATPase cycle.We measured myosin Va head stiffness with a modified three-bead trapping assay. One bead of the dumbbell was attached end-on to the actin filament to reduce compliance of this link. To determine myosin Va stiffness we applied ramp-shaped movements to the stage and followed the position of the end-on attached bead. When a myosin Va head bound to the dumbbell, the dumbbell started to follow the stage movement, however, with reduced amplitude. The reduced amplitude we attributed to elastic distortion of the myosin Va head, elastic extension of the actin-bead link, and compliance between the myosin Va molecule and the nitrocellulose coated third bead. Compliances of the actin-bead links were determined according to Smith et al. (Biophys J 2001) and by force-extension curves.Coinciding with the substep in myosin Va binding events bead movement suddenly followed the imposed stage movement more closely than before the substep, indicating suddenly reduced elastic extension of myosin Va. Quantitative analysis suggests a stiffness of the chicken myosin Va head domain of about 0.2pN/nm before the substep and a 2.5 to 3-fold increase in stiffness with the substep.