Coupling of ATPase activity and motility in smooth muscle myosin is mediated by the regulatory light chain.

Smooth muscle myosin acts as a molecular motor only if the regulatory light chain (RLC) is phosphorylated. This subunit can be removed from myosin by a novel method involving the use of trifluoperazine. The motility of RLC-deficient myosin is very slow, but native properties are restored when RLC is rebound. Truncating 6 residues from the COOH terminus of the RLC had no effect on phosphorylated myosin's motor properties, while removal of the last 12 residues reduced velocity by approximately 30%. Very slow movement was observed once 26 residues were deleted, or with myosin containing only the COOH-terminal RLC domain. These two mutants thus mimicked the behavior of RLC-deficient myosin, with the important difference that the mutant myosins were monodisperse when assayed by sedimentation velocity and electron microscopy. The decreased motility therefore cannot be caused by aggregation. A common feature of RLC-deficient myosin and the mutant myosins that moved actin slowly was an increased myosin ATPase compared with dephosphorylated myosin, and a lower actin-activated ATPase than obtained with phosphorylated myosin. These results suggest that the COOH-terminal portion of an intact RLC is involved in interactions that regulate myosin's "on-off" switch, both in terms of completely inhibiting and completely activating the molecule.

Chimeric regulatory light chains as probes of smooth muscle myosin function.

Functional domains of the smooth muscle regulatory light chain (LC) were identified from the assembly and motor properties of smooth muscle myosin containing chimeric LCs, in which the N- and C-terminal halves of the smooth and skeletal LCs were interchanged. A C-terminal domain was also expressed. The affinity of these LCs for the smooth muscle myosin heavy chain (HC) is: wild-type LC > N-skeletal/C-smooth > N-smooth/C-skeletal approximately skeletal LC >> C-terminal domain. The C-terminal half of the LC thus contains an isoform-specific HC binding site, but the two halves of the LC must interact for tight binding. Smooth muscle myosin containing chimeric or skeletal LCs can no longer assume the folded monomeric conformation, suggesting that control of assembly involves both halves of the LC. Dephosphorylation/phosphorylation of the N-skeletal/C-smooth chimera nonetheless regulates the ability of smooth muscle myosin to move actin. Myosin containing phosphorylated N-smooth/C-skeletal or skeletal LCs, in contrast, is locked in the "off" state. Interactions between the stronger binding C-terminal domain of the LC and the HC are therefore primarily responsible for the regulatory capabilities of this subunit. Localization of the regulatory LC at the head/rod junction by electron microscopy establishes that phosphorylation-induced changes must be transmitted over 10 nm within the head for product release to be enhanced.