Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin.
We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.
Nelson
Wendy D
WD
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Blakely
Sarah E
SE
Nesmelov
Yuri E
YE
Thomas
David D
DD
D000818:Animals; D002645:Chickens / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D024745:Smooth Muscle Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013113:Spin Labels
D013113:Spin Labels; D024745:Smooth Muscle Myosins