The Molecular Mechanism Of Phosphoryla-tion Regulation Of Smooth Muscle Myosin

Smooth muscle myosin (SmM), the major component of smooth muscle thick filaments, is an important molecular motor. SmM is able to convert the energy from ATP hydrolysis into mechanical movement along actin thin filaments, causing the contraction of smooth muscle.SmM is composed of two heavy chains, two essential light chains (ELC), and two regulatory light chains (RLC). The heavy chain contains an N-terminal motor domain, followed by ELC and RLC binding sites, and an C-terminal coiled coil tail. The motor function of SmM is regulated by phosphorylation of RLC. In unphosphorylated state, SmM motor function is inhibited and has very low ATPase activity; upon RLC phosphorylation, SmM motor function is stimulated to maximal level and has high ATPase activity. It is now widely accepted that unphosphorylated SmM is in a folded inhibited state and phosphorylation of RLC disrupts above interactions and induces SmM to form an extended activated state. Since the phophorylation site is located in RLC, which is far from the ATPase site, a critical question is how RLC phosphorylation regulates the motor function.Firstly, We investigated the role of double head in SmM phosphorylation regulation. The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions,thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM. This new model can explain almost all the published date of SmM.We then investigated the interaction between the head and tail of SmM on the inhibition of motor function in unphosphorylated state. A number of studies indicate that the head-tail interaction between unphosphorylated SmM is essential for the inhibitory state. However, the underlined molecular mechanism remains to be determined. To determine the role of tail region on the inhibitory state of SmM, we substituted different regions of the coiled-coil tail with Leu zipper and identified two conserved tail regions which are rich in acidic residues critical for the inhibitory state of SmM. Moreover, we identified the critical residues in these two tail regions for the inhibitory state of SmM. Based on these results, we proposed that, in unphosphorylated state, the conserved acidic residues in the tail interact with the conserved basic residues in the head of SmM. Consistently, we found that the highly conserved basic residue R406is essential for the inhibitory state of unphorylated SmM.In summary, we investigated the role of double-headed structure on the phosphorylation-regulation of SmM motor function and demonstrated that the conserved acidic residues in the tail and basic residues in the head are critical for the inhibition of motor function in unphosphorylated SmM. This study not only revealed the molecular mechanism of SmM regulation by phosphorylation, but also shed light on the molecular machinery that underlies the regulation of molecular motor proteins.