Molecular dynamics simulations reveal a disorder-to-order transition upon phosphorylation of smooth muscle myosin

¹ University of Minnesota
² University of Minnesota Medical School

^* To whom correspondence should be addressed. E-mail: ddt{at}umn.edu.

Submitted on August 23, 2006
Revised on October 19, 2006
Accepted on 11 January 2007

	Abstract

We have performed molecular dynamics simulations of the phosphorylated (at S19) and unphosphorylated 25-residue N-terminal phosphorylation domain of the regulatory light chain (RLC) of smooth muscle myosin, in order to provide insight into the structural basis of regulation. This domain does not appear in any crystal structure, so these simulations were combined with site-directed spin labeling to define its structure and dynamics. Simulations were carried out in explicit water at 310 K, starting with an ideal -helix. In the absence of phosphorylation, large portions of the domain (residues S2 to K11 and R16 through Y21) were metastable throughout the simulation, undergoing rapid transitions among -helix, -helix, and turn, while residues K12 to Q15 remain highly disordered, displaying a turn motif from 1 to 22.5 ns and a random coil pattern from 22.5 to 50 ns. Phosphorylation increased -helical order dramatically in residues K11 to A17 but caused relatively little change in the immediate vicinity of the phosphorylation site (S19). Phosphorylation also increased the overall dynamic stability, as evidenced by smaller temporal fluctuations in the RMSD. These results on the isolated phosphorylation domain, predicting a disorder-to-order transition induced by phosphorylation, are remarkably consistent with published experimental data involving site-directed spin labeling of the intact RLC bound to the two-headed heavy meromyosin. The simulations provide new insight into structural details not revealed by experiment, allowing us to propose a refined model for the mechanism by which phosphorylation affects the N-terminal domain of the RLC of smooth muscle myosin.

Key Words: EPR, metastable, pi-helix, regulation, regulatory light chain, spin label