TR-FRET Experiments and MD Simulations Resolve Structural States of Smooth Muscle Regulatory Light Chain

Abstract

We have combined time-resolved fluorescence resonance energy transfer (TR-FRET) and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of chicken gizzard smooth muscle regulatory light chain (RLC) bound to smooth muscle myosin. The 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, 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 RLCs, with and without phosphorylation at S19, were reconstituted into myosin S1. Time-resolved FRET identified two structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. Phosphorylation shifts the equilibrium toward the open state by 20-30%. Molecular dynamics simulations, guided by these and previous spectroscopic studies, confirm the existence of the open and closed structural states and produce atomic resolution models of these states. In the closed state, the PD interacts with the surface of the C-terminal lobe. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is more mobile. Phosphorylation stabilizes the open state by forming a specific salt bridge between R16 and phosphorylated S19. This conformational shift is consistent with a mechanism of regulation that catalyzes large structural changes within myosin at a low energetic cost. This work was supported by grants from NIH (AR32961, AR07612) and the Minnesota Supercomputing Institute. We thank Igor Negrashov for excellent technical assistance.