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ABSTRACT
We have performed molecular dynamics simulations of the phosphorylated (at S-19) and the unphosphorylated 25-residue N-terminal phosphorylation domain of the regulatory light chain (RLC) of smooth muscle myosin 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 α-helix. In the absence of phosphorylation, large portions of the domain (residues S-2 to K-11 and R-16 through Y-21) were metastable throughout the simulation, undergoing rapid transitions among α-helix, π-helix, and turn, whereas residues K-12 to Q-15 remained 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 α-helical order dramatically in residues K-11 to A-17 but caused relatively little change in the immediate vicinity of the phosphorylation site (S-19). Phosphorylation also increased the overall dynamic stability, as evidenced by smaller temporal fluctuations in the root mean-square deviation. 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.
INTRODUCTION
Each muscle myosin (isoform II) molecule contains two heavy chains, and two pairs of associated calmodulin-like proteins called the essential light chain (ELC) and the regulatory light chain (RLC). The heavy chain is comprised of an N-terminal globular catalytic domain that contains both actin-binding and ATP-hydrolysis activities and an extended C-terminal a-helix that serves in dimerization and filament formation. Both the ELC and RLC are bound to this helical heavy chain region proximal to the N-terminus to form the light-chain domain (1) (Fig. 1).
In smooth muscle, phosphorylation of Thr-18 or Ser-19 on RLC by the Ca^sup 2+^/calmodulin-dependent myosin light chain kinase (MLCK) is required for activation of muscle contraction. The unphosphorylated state of smooth muscle myosin (SMM) has negligible catalytic activity, but phosphorylation produces...