Neck Length and Processivity of Myosin V

Abstract

Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 M or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.The myosin superfamily consists of at least 18 classes of actin-dependent molecular motors (1). Class V myosins transport cargos such as endoplasmic reticulum in neurons, melanosomes in melanocytes, and mRNA in yeast (2). Similar to other myosins, they are composed of a head that binds ATP and actin and a neck region consisting of calmodulin (CaM) 1 molecules bound to an ␣-helical segment of the myosin heavy chain. The C-terminal tail domain of myosin V has coiled-coil forming motifs that dimerize and create two-headed molecules but do not self-associate into filamentous structures. The IQ motifs of the heavy chain, implicated in the binding of CaM or light chain subunits, have the consensus sequence, IQXXXRGXXXR, where X is any amino acid (3). The neck of mouse myosin V has six IQ motifs, each of which binds CaM, making its neck longer than that of most myosins (4). The six IQ motifs of all myosin V superfamily members are separated by 25-23-25-23-25 amino acids.The well studied myosin II class molecules that power muscle contraction and participate in cytokinesis in nonmuscle cells polymerize into filaments that are interdigitated with actin filaments. Kinetic and mechanical studies have demonstrated that these myosins interact only transiently with actin and typically spend greater than 95% of their kinetic cycle detached from actin. The term "low duty cycle" motor has been used to describe this behavior adopted to allow the sliding of actin filaments past myosin filaments to be unimpede...

The structure of the myosin VI motor reveals the mechanism of directionality reversal

“…The lever arm further amplifies the motions of the converter domain into large directed movements. Consistent with the lever arm hypothesis, the stroke size has been shown to be proportional to the lever arm length 8,9,10 . In the absence of actin, ATP hydrolysis occurs, but product release is slow, thus trapping the lever arm in a primed or pre-powerstroke position.…”

Step-Size Is Determined by Neck Length in Myosin V

“…The presence of strain-dependent intrahead gaiting of the product release rates from wild-type myosin V likely contributes to the processivity (2,22). The rate of movement of the 8IQ-HMM mutants in single-molecule TIRF assays at 2 mM ATP was slower than that of 6IQ-HMM despite the longer step-size in the former (8). This may reflect a loss of strain-dependent acceleration of the rate of ADP release from the trailing head in this mutant (26).…”

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

“…Each myosin family member has a variable number of IQ motifs that form the light chain-binding sites for calmodulin that comprise the lever arm (20). Myosin V produces long working strokes with its long lever, and truncations produce working strokes in proportion to the lever arm length (8,9). Myosin X has a short, three-IQ lever arm, which is half of the six-IQ lever arm of the canonically processive myosin V. Part of this difference may be compensated by a SAH motif (13) that could extend the lever arm.…”

“…4A). Such an approach was previously used to break the myosin V lever arm after the second IQ domain (9). The linker of choice was (GSG) 2 as it has been previously used on anchoring domains on myosin VI (21).…”