Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain.
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 approximately 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.
Kast
David
D
Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Espinoza-Fonseca
L Michel
LM
Yi
Christina
C
Thomas
David D
DD
D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D024745:Smooth Muscle Myosins / Q000737:chemistry* / Q000378:metabolism*
D024745:Smooth Muscle Myosins
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