We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by ∼20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle. fluorescence resonance energy transfer | FRET | molecular dynamics | molecular dynamics simulation | time-resolved fluorescence S mooth muscle myosin (SMM) is a member of the myosin superfamily of motor proteins, which use chemical energy from ATP hydrolysis to perform mechanical work on actin. Motor properties of myosin are activated by Ca 2þ . In skeletal muscle, Ca 2þ moves actin-bound inhibitory proteins to allow active myosin-actin interaction. In smooth muscle, activation requires phosphorylation of regulatory light chain (RLC) (Fig. 1), which is distant from the myosin active site on the catalytic domain (Fig. 1). Unphosphorylated SMM is auto-inhibited by interactions between the two catalytic domains (1, 2) that are relieved by RLC phosphorylation (3, 4). Although regulation requires both heads of myosin (5-8), phosphorylation of one RLC is sufficient to activate both heads (9, 10).The structural mechanism by which phosphorylation of RLC at S19 activates SMM is a mystery, primarily because there is no highresolution structure of any myosin that contains the N-terminal 24 residues of RLC (Fig. 1). Site-directed spin labeling demonstrated that this N-terminal segment acts as a coherent domain in response to phosphorylation, so it is now referred to as the phosphorylation domain (PD) (11). Removal of PD abolishes regulation, but charge replacement or deletion of PD causes only partial activ...

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 alpha-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 alpha-helix, pi-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 alpha-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.

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