Atomic-Level Visualization of Smooth Muscle Activation by Phosphorylation of the Myosin Regulatory Light Chain

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We have engineered site-directed probe pairs in the regulatory light chain (RLC) for exchange into smooth muscle heavy meromyosin (HMM) and subfragment-1 (S1), in order to examine the phosphorylation-induced changes in RLC structural states using time-resolved FRET (TR-FRET). Phosphorylation of the RLC is required for activation of contraction in smooth muscle and modulates force in striated muscle. Force development in smooth muscle is triggered by phosphorylation of the RLC's N-terminal phosphorylation domain (PD) at Ser19, alleviating inhibitory interactions between the two heads. Since crystal structures of the RLC lack the PD, the mechanism by which RLC phosphorylation allosterically triggers disruption of HMM auto-inhibition remains unresolved. Our site-directed FRET probe pairs resolved intramolecular atomic distance measurements between the RLC core and the dynamic PD. Smooth muscle RLC assumes two structural states in both states of activation: phosphorylation shifts the equilibrium of these two structural states from primarily occupying the compact closed state to favoring the extended open state of the PD, relative to the RLC core. Compared to single-headed S1 (Kast et al., PNAS., 2010), the open state RLC conformation of unphosphorylated HMM is more compact, presumably due to head-head or RLC-RLC interactions. All-atom MD simulations corroborate our TR-FRET studies, and reveal specific salt-bridges that stabilize each structural state. The hypotheses generated from our MD simulations are being tested experimentally by TR-FRET via site-directed mutagenesis to manipulate charge. These studies offer the first atomic-resolution insight into the structural dynamics of RLC phosphorylation, and a similar approach should be applicable to striated muscle. Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR0507220, T32 AR007612).