Temperature Dependence of Nucleotide Association and Kinetic Characterization of Myo1b

Abstract

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. It is one of the best characterized myosin-I isoforms and appears to have unique biochemical properties tuned for tension sensing or tension maintenance. We determined the key biochemical rate constants that define the actomyo1b ATPase cycle at 37 °C and measured the temperature dependence of ATP binding, ADP release, and the transition from a nucleotide inaccessible state to a nucleotide accessible state (kalpha). The rate of ATP binding is highly temperature sensitive, with an Arrhenius activation energy 2-3 fold greater than other characterized myosins (e.g, myosin-II and myosin-V). ATP hydrolysis is fast, and phosphate release is slow and rate limiting with an actin dependence that is nearly identical to the steady-state ATPase parameters (V max and K ATPase ). ADP release is not as temperature dependent as ATP binding. The rates and temperature dependence of ADP release are similar to kalpha suggesting a similar structural change is responsible for both transitions. We calculate a duty ratio of 0.08 based on the biochemical kinetics. However, this duty ratio is likely to be highly sensitive to strain.Myosin-Is are the single-headed, low-molecular-weight members of the myosin superfamily that are proposed to link cellular membranes with the actin cytoskeleton. Myosin-I isoforms bind phosphoinositides directly (1) and function in several important cellular processes, including membrane retraction, macropinocytosis, phagocytosis, membrane trafficking, cellcell adhesion, and mechanical signal transduction (2-8).The biochemical mechanisms of long-tail and short-tail myosin-I isoforms have been studied (9-12), with myo1b being the best characterized short-tail isoform (13)(14)(15). The myo1b ATPase mechanism (Scheme 1) is notable in that (a) the maximum rate of ATP binding and population of the myo1b IQ weakly-bound states is > 30-fold slower than myosin-II, (b) nucleotide-free myo1b is in equilibrium between a state that binds nucleotide (AM′) and a state that does not bind nucleotide (AM), (c) the rate of transition between AM and AM′ (k +α ) states is similar to the rate of ADP release (k +5 ′), and (d) ADP release is slow and is accompanied by a rotation of the lever arm.We proposed that the strikingly slow actomyo1b ATPase rate constants are a property of all short-tail myosin-I isoforms (11,16). However, it has been shown recently that short-tailed Dictyostelium myosin-IE (not to be confused with vertebrate long-tail myo1e (11)) has kinetic rate constants that are substantially faster than vertebrate short-tail isoforms (12). Because of † EMO was supported by grants from the National Institutes of Health (GM57247 and AR051174). JHL was supported by a training grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR053461). *Corresponding author: E. Michael Ostap, Department of Physiology, University of Pe...

Myosin IC generates power over a range of loads via a new tension-sensing mechanism

“…The ratio of affinities (K 10 ∕K 6 ¼ K DA ∕K A ) has been termed the coupling constant (22,23), and the calculated value of 5.5 is similar to previously published values for myo1b (28,29); however, it is larger than a previously reported value for myo1c (10). Based on the solution kinetics, ADP release is expected to be rate limiting for actomyosin detachment at saturating ATP concentrations; however, a slower transition (likely associated with phosphate release) (16,19) limits the rate of the overall ATPase cycle. The effective duty ratio of myo1c 3IQ at 100 μM actin and saturating ATP concentrations is 0.11.…”

“…The rate constants that define the myo1c actin-activated ATPase pathway in solution are very similar to myo1b (10,12,16,19), a closely related myosin-I isoform that has actin-detachment kinetics that are exquisitely sensitive to tension (20). Myo1b is suited to function as a tension-sensitive anchor, transforming from a low duty-ratio motor to a high duty ratio-motor with forces resisting its working stroke >0.5 pN.…”

Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear

“…Myosin 1b has been shown to have an unusually marked temperature dependence (29). We made preliminary studies of the temperature dependence of some of the key steps in Myo1c 1IQ and found that Myo1c shares some of the temperature dependence of Myo1b but is also quite distinct (see SI Text and Fig.…”

Control of myosin-I force sensing by alternative splicing

“…4) (7,13). Experiments were performed in the presence of 50 μM ATP to ensure that the rate of ATP binding did not limit the rate of detachment (7,17). Long attachment durations at higher forces are apparent in the time traces of all four myo1b proteins, but it is clear that the shorter isoforms have shorter attachment durations (Fig.…”