Abstract
Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. However, the head is kept in close proximity to actin due to the tethering role of loop 2/large unique insertion of myosin IX. There is enough freedom for the myosin head to find the next location of the binding site along with the actin filament before complete dissociation from the filament. After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place.
Highlights
A critical issue is that myosin IXb is a single-headed myosin yet moves processively on actin filaments
We expressed and purified the M9bIQ4 construct (Fig. 1A) that contained the entire head domain and the light chain binding neck domain, because this shows the processive movement on actin filament, and one can avoid the potential complexity arising from the presence of the tail domain containing the Rho-binding GTPase-activating protein
The results indicate that M1⁄7ATP state for myosin IX has much higher binding affinity than other myosins, in which the M1⁄7ATP state is a weak actin binding state [32,33,34], including processive myosin VI [35]
Summary
Reagents and Proteins—2Ј-Deoxy-mant-ATP (dmantATP) was kindly provided by Dr Howard D. The recombinant myosin IXb was expressed as described [5]. 2 mM ATP was added and incubated for 5 min at room temperature. For the binding assay of the M1⁄7ADP1⁄7P state, 0.1 mM ADP and 1 mM Vi were incubated with M9bIQ4 for 1 h on ice, and various concentrations of actin were incubated with the ternary complex of M9bIQ41⁄7ADP1⁄7Vi for 5 min at room temperature. To determine the bound M9bIQ4 containing ADP1⁄7Vi, the amount of [3H]ADP trapped in acto-M9bIQ4 was measured. The concentration of bound M9bIQ4 in dissolved pellet was determined by densitometry of an SDS-polyacrylamide gel, and the percentage of trapped [3H]ADP in the M9bIQ4 was calculated. Samples of M9bIQ4 or acto-M9bIQ4 were mixed with an equal volume of [32P]ATP in buffer. Kinetic modeling and simulation were performed using STELLA software version 8.1.1 (Isee Systems)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
