Abstract
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.
Highlights
Functional domainsof the smooth muscle regulatory Phosphorylation-dependent regulatiounndoubtedldyelight chain (LC)were identified from the assembly and pends on the appropriate interactions between both heavy motor properties of smooth muscle myosin containing and light chains
The regulatory capabilities of the light chain primarily depend on a native C-terminadl omain, whereas the ability to assume the folded monomeric conformation requires interactions involving both halves of the light chain
Light ChuinlHeavy Chain Binding-Both halves of the light chain contain isoform specific heavy chain binding sites, but sequences present in the C-terminal domain are more critical since the N-skeletal/C-smooth chimera has only a lO-fold lower affinity for the heavy chain than thenative light chain, whereas the N-smooth/C-skeletal chimera has a 100-fold lower affinity
Summary
Functional domainsof the smooth muscle regulatory Phosphorylation-dependent regulatiounndoubtedldyelight chain (LC)were identified from the assembly and pends on the appropriate interactions between both heavy motor properties of smooth muscle myosin containing and light chains. The hybrid myosins obtained by light chain exchange were increased to 80% for both the N-smooth/C-skeletal chimera assayed for their ability to undergo the extended-to-folded (Fig. 5B,P)and skeletal light chain(data not shown).
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