Abstract
The emerging view of smooth/nonmuscle myosin regulation suggests that the attainment of the completely inhibited state requires numerous weak interactions between components of the two heads and the myosin rod. To further examine the nature of the structural requirements for regulation, we engineered smooth muscle heavy meromyosin molecules that contained one complete head and truncations of the second head. These truncations eliminated the motor domain but retained two, one, or no light chains. All constructs contained 37 heptads of rod sequence. None of the truncated constructs displayed complete regulation of both ATPase and motility, reinforcing the idea that interactions between motor domains are necessary for complete regulation. Surprisingly, the rate of ADP release was slowed by regulatory light chain dephosphorylation of the truncated construct that contained all four light chains and one motor domain. These data suggest that there is a second step (ADP release) in the smooth muscle myosin-actin-activated ATPase cycle that is modulated by regulatory light chain phosphorylation. This may be part of the mechanism underlying "latch" in smooth muscle.
Highlights
The regulation of vertebrate smooth and nonmuscle myosin II is based on reversible phosphorylation of one of the two myosin light chains, referred to as the regulatory light chain (RLC)1 (1–3)
We have suggested that smooth muscle-specific sequences near the motor domain may contribute to the proper positioning of the light chains that is critical to facilitate the RLC-rod-head interactions necessary for achieving a completely off state in the absence of RLC phosphorylation (2)
To ascertain whether interactions between the light chain binding domains can confer a high degree of regulation, we constructed a series of asymmetric HMMs and assessed the degree of regulation conferred on these constructs by RLC phosphorylation
Summary
To further examine the nature of the structural requirements for regulation, we engineered smooth muscle heavy meromyosin molecules that contained one complete head and truncations of the second head These truncations eliminated the motor domain but retained two, one, or no light chains. A number of lines of evidence have led to the proposal that there must be interactions between the two light chain binding domains of the heads (3, 7) If these are the most important interactions for regulation, it might be possible to preserve regulation by eliminating one of the two smooth muscle myosin motor domains, while retaining one or both of the light chain binding domains of that head, as well as the complete second head. To ascertain whether interactions between the light chain binding domains can confer a high degree of regulation, we constructed a series of asymmetric HMMs and assessed the degree of regulation conferred on these constructs by RLC phosphorylation
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