The gated gait of the processive molecular motor, myosin V

Abstract

Class V myosins are actin-based molecular motors involved in vesicular and organellar transport. Single myosin V molecules move processively along F-actin, taking several 36-nm steps for each diffusional encounter. Here we have measured the mechanical interactions between mouse brain myosin V and rabbit skeletal F-actin. The working stroke produced by a myosin V head is approximately 25 nm, consisting of two separate mechanical phases (20 + 5 nm). We show that there are preferred myosin binding positions (target zones) every 36 nm along the actin filament, and propose that the 36-nm steps of the double-headed motor are a combination of the working stroke (25 nm) of the bound head and a biased, thermally driven diffusive movement (11 nm) of the free head onto the next target zone. The second phase of the working stroke (5 nm) acts as a gate - like an escapement in a clock, coordinating the ATPase cycles of the two myosin V heads. This mechanism increases processivity and enables a single myosin V molecule to travel distances of several hundred nanometres along the actin filament.

Step-Size Is Determined by Neck Length in Myosin V

“…For the 6IQ-HMM, this movement corresponded to about 74 nm, but the center of mass of the molecule has only moved one-half of this distance (37 nm). Thus, the step-sizes measured in the FIONA assay are consistent with those measured by optical trapping where the center of mass movement of the bead is measured (2,7). In both the optical trapping studies where myosin V molecules were moving against load and in the FIONA system where there was no load, the step-size of 6IQ myosin V is matched with the helical repeat of the actin filament (8)(9)(10).…”

“…The rate was fastest for 6IQ-HMM and about 2-fold slower for 4IQ-HMM. In optical trapping assays and TIRF single molecule movement assays, at limiting ATP concentrations, the rate of stepping of the 6IQ-HMM or intact myosin V molecules is consistent with the second-order rate constant of ATP binding to actomyosin V S1, as measured by transient kinetics (2,7). It is possible that the slower stepping mutants in this study have a slower rate of ATP binding due to distortion of the molecules caused by intramolecular strain as the two heads bind at different azimuthal angles.…”

“…The long neck region, which most likely functions as a lever-arm to amplify small nucleotidedependent structural changes in the motor domain, permits myosin V to take steps of an average distance of ∼36 nm, coinciding with the length of the half-pitch of an actin filament (2,7). This step consists of a working stroke (defined as the distance myosin V translocates actin in a single movement of the lever-arm), followed by a diffusive search by the new lead head for a suitable binding site on actin.…”

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

“…19,20 Myosin V walks toward the plus end of actin by taking ∼37 nm steps per ATP hydrolyzed. 21,22 Myosin V transports a wide variety of cargo including pigment granules, membranous organelles and secretory vesicles in vertebrates, and mRNA in yeast. Functional defects in this protein cause neurological diseases and pigmentation in mice and humans.…”

Kinesin motors as molecular machines

“…(29) ATP hydrolyzed. (33,34) Thermal fluctuations are expected to play an important role in the motor mechanism, either by driving diffusional movements of the motor to its next binding position (35) or by promoting diffusive structural changes that drive force-producing conformational changes. (30) The structural elements that undergo strain have not been identified with certainty for any molecular motor, but are likely to have spring-like or elastic properties that allow them to extend or rotate, then recoil back into their original conformation.…”