Activation of smooth muscle myosin (SMM) requires phosphorylation of the myosin's regulatory light chain (RLC) to relieve autoinhibitory head-head interactions, but the structural basis of this mechanism in unknown. There are no crystal structures of any fragment of SMM, and there are no crystal structures of any RLC that contains the N-terminal 24 amino acids required for phosphorylation. Site-directed spin labeling of this N-terminal segment, referred to as the phosphorylation domain (PD), showed that phosphorylation increases α-helicity, mobility and solvent accessibility of the PD. A model emerged, where the unphosphorylated RLC is compact with a disordered PD, and phosphorylation causes the PD extend away from the RLC while inducing helical ordering. The goal of this research is to test the hypothesis that the PD functions as a structural switch that changes the structure of RLC upon phosphorylation, and to define these structural changes in atomic detail.
Complementary fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations were performed to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC). MD simulations on the isolated PD reveal disorder-to-order transition, where residues K11-Q15 are disordered in the unphosphorylated PD but completely α-helical in the phosphorylated PD. A salt bridge formed between R16 and the phosphorylated S19 promotes ordering by stabilizing α-helicity and reducing conformational fluctuations. Consequently, this disorder-to-order transition is regulated by delicate balance between enthalpy and entropy. To elucidate the structural changes of the PD in context with the RLC bound to smooth muscle myosin, 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 S1. Time-resolved FRET demonstrated two simultaneously resolved structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mole fraction by 23%. Molecular dynamics simulations agree with FRET data in remarkable detail, supporting the coexistence of two structural states, with phosphorylation shifting the system toward a more open and mobile structure. This agreement between experiment and simulation validates the additional structural details provided by the MD simulations: In the closed state, the PD is bent onto the surface of the C-terminal lobe, stabilized by two specific interdomain salt bridges. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by a specific intradomain salt bridge. The closed and open states are also present in phosphorylated HMM, while unphosphorylated HMM possess the closed state an intermediate distance distribution. Phosphorylation forces the PD to adopt S1-like states without increasing the mean separation of the two myosin heads. The result is a vivid atomic-resolution model of the molecular mechanism by which phosphorylation activates smooth muscle.