Role of the Essential Light Chain in the Activation of Smooth Muscle Myosin by Regulatory Light Chain Phosphorylation

Abstract

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at ser19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. The mechanism by which ser19 phosphorylation disrupts these head-head interactions is unknown. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837-854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Based on this model, placement of the regulatory domain at this locus might alter relative motions between the ELC and the RLC and thereby disrupt the inhibited state. Here we derive an atomic model based on this hypothesis for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). We use a homology model for the structure of the RLC and a largely α-helical structure for the regulatory domain. This model predicts a set of specific interactions between the regulatory domain and both the myosin HC and the ELC. Site directed mutagenesis combined with ATPase and motility assays was used to show that interactions between the highly basic N-terminus of the RLC with acidic residues on helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin. These sites are well conserved in all the myosin II ELC sequences examined despite the lack of known interacting partners and when substituted, are usually substituted with aspartic acid. Supported by NIH grants AR47421, AR53975 and HL38113 and RR12255.