Mechanism of Nucleotide Binding to Actomyosin VI

Abstract

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds singleheaded myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K 1 ‫؍‬ 5.6 mM) followed by isomerization (k ؉2 ‫؍‬ 176 s ؊1 ) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is ϳfive times greater than for ATP binding because of a higher affinity in the collision complex (1/K 5b ‫؍‬ 2.2 mM) and faster isomerization rate constant (k ؉5a ‫؍‬ 366 s ؊1 ). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.The myosin family of molecular motors constitutes a large gene family of proteins that couple the energy from ATP binding, hydrolysis, and product release to force generation along actin filaments (1). At least 18 classes (Classes I-XVIII) make up the family (2). All possess a highly conserved catalytic motor domain (referred to as "head") that binds actin, hydrolyzes ATP, and performs the mechanical work. In nonmuscle cells, cytoplasmic myosins are required for various cellular processes including membrane and RNA trafficking, cell migration, and cytokinesis (3).The nucleotide bound to myosin dictates the affinity for actin filaments (1). In the absence of nucleotide or with bound ADP, myosin binds actin filaments with high (Ͻ1 M) affinity; these states are referred to as "strongly bound states." With ATP or the hydrolysis products, ADP-P i , bound, myosin binds actin with low affinity and rapidly attaches and detaches from the actin filament on a submicrosecond timescale; these nucleotide states are called "weakly bound states." The strong binding states are force-bearing intermediates, but the weak binding states are not. The fraction of the total ATPase cycle time spent strongly bound to actin is called the duty ratio. Because under physiological nucleotide concentrations ATP binding is largely favored over ADP binding, ADP release limits the lifetime of the strongly bound actomyosin states and dictates the duty ratio.Ther...

The power stroke of myosin VI and the basis of reverse directionality

“…At low [ATP], kinetics are in reasonable agreement with previous kinetic studies of myosin VI (11,27,28). M6CD and M6PI have slightly altered kinetics at saturating [ATP], suggesting that truncation may have a small effect on ADP release but not on ATP binding.…”

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

“…These constructs travel for much shorter distances than observed for wild type on bundles. This reduced run length may be the result of a disruption of a sensitive gating mechanism where the ATPase cycles of the two heads are regulated by internal stress as is common for processive myosins (23,24). Consistent with this idea, we find that these swivels increase the actin-activated ATPase V max by ϳ50%, suggesting that gating is partially disrupted due to the increased flexibility of the swivels (supplemental Fig.…”

Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor

“…7A shows the k obs values of single exponential fits to the pyrene fluorescence transients recorded on rapidly mixing 0.15 M pyrene-actin plus 0.2 M mX-S1 with a nucleotide mixture consisting of 200 M ATP plus the indicated ADP concentrations (pre-mixing concentrations indicated). As discussed in a recent detailed investigation of the ADP interaction of pyrene-actomyosin VI (29), double exponential transients would be expected in this reaction, but the two phases were not resolved in our experiments. Nevertheless, the initial (zero [ADP]) k obs decreased to half at 30 M ADP (Fig.…”

“…Such an alteration in the mechanism would not change the ATP-related kinetic behavior of the enzyme.) The absence of a marked acceleration of the ADP release step by actin is not without precedent in the myosin superfamily; nonmuscle myosin IIA (21), IIB (17), and myosin VI (29,40) have been shown previously to exhibit a similar behavior.…”