Modeling The Mechanics and Enzymatic Activities of Doubly, Singly and Unphosphorylated Smooth Muscle Myosin

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Smooth muscle myosin II has two heads, each capable of ATP hydrolysis and actin binding. However, both heads are required for maximal force and motion generation (Tyska et al., 1999), where one head, by weakly interacting with actin, may serve as a “tether and guide,” to optimize the second head's mechanical performance (Kad et al., 2003). In addition to these non-equivalent mechanical roles, an asymmetric head-head interaction inhibits the mechanical and ATPase activities of unphosphorylated (0P) smooth muscle myosin where one head's (blocked head) actin-binding domain associates with the converter region of the second head (free head). How does the equilibrium between active and inhibited states depend on myosin's phosphorylation state and specifically when only one of its two heads is phosphorylated? Once fully active, how quickly do the heads exchange their mechanical roles? We have addressed these questions in a multistate kinetic and mechanical model.Based on single ATP turnover and steady-state ATPase data for fully active doubly phosphorylated (2P) smooth muscle myosin (Ellison et al. 2000, Rovner et al. 2006), we predict that, surprisingly, the two heads exchange their mechanical roles (i.e. weak vs. strong-binding head) slowly - at a rate of 0.1-0.3s^-1. By comparing ATPase and single molecule mechanical data (step size, attachment time, force-velocity) from fully active (2P), inhibited (0P), and smooth muscle myosin with only one head phosphorylated (1P), we predict that an equilibrium between active and inhibited conformations, which is phosphorylation-state dependent, can explain the enzymatic and mechanical data for all three phosphorylation states (2P, 1P and 0P). However, an alternative model can be proposed where the active and inhibited state equilibrium in the 1P state depends on the mechanical role of the phosphorylated head.