Phosphorylation-Induced Structural Changes in Smooth Muscle Regulatory Light Chain

Abstract

We are using site-directed spectroscopic labeling, fluorescence, and molecular dynamics simulations to define the phosphorylation-induced structural transition in smooth muscle myosin regulatory light chain (RLC). Smooth muscle is activated through phosphorylation of Ser 19 on RLC, but the N-terminal 24 amino acids of RLC do not appear in any crystal structure. EPR experiments (Nelson et al., 2005) have shown that phosphorylation induces a disorder-to-order transition within the N-terminal phosphorylation domain of the RLC, in which increased helical ordering relieves inhibitory head-head interactions. To define this structural change in atomic detail, we are combining molecular dynamics simulations with spectroscopic distance constraints. Simulations on the unphosphorylated 25-residue N-terminal fragment of the RLC reveal a disordered region in T9-K12, while the phosphorylated N-terminal domain maintains strong α-helicity over the same residues. This disorder-to-order transition is regulated by delicate balance between enthalpy and entropy involving R16. The same disorder-to-order transition has been observed by both simulations extended to include the entire RLC in complex with a portion of the myosin heavy chain. Furthermore, we have employed FRET distance measurements on di-Cys mutant RLC exchanged onto S1 and HMM, to provide geometric constraints for the simulations. This allows determination of both structure and dynamics of the regulatory domain in the absence and presence of phosphorylation. This work was supported by grants from NIH (AR32961, AR07612) and the Minnesota Supercomputing Institute. We thank Igor Negrashov for excellent technical assistance.