Calponin binds to the 20-kilodalton regulatory light chain of myosin.
Calponin (CaP) is a 34 kDa smooth muscle-specific protein that has been implicated in regulation of smooth muscle contractility. Two CaP binding sites on smooth muscle myosin rod have been recently described [Szymanski and Tao (1997) J.Biol.Chem. 272, 11142-11146]. We used a combination of cosedimentation, overlay, and fluorescence assays to determine the interaction between CaP and both subfragment 1 of myosin and isolated 20 kDa regulatory light chain of myosin (RLC). Subfragment 1, which was generated by cleavage of myosin with Staphylococcus aureus protease (myosin S1SA) inhibits cosedimentation of CaP with myosin filaments. Fluorescence assay showed that CaP labeled with fluorescent label (DAN-CaP) interacts with myosin S1SA in solution via a single class of binding sites. The binding constant (kaff) of this interaction at 50 mM NaCl is (2. 1 +/- 0.2) x 10(6) M-1 (n = 3). The interaction between DAN-CaP and myosin S1SA depends on ionic strength, and the EC50 of inhibition of this interaction occurs at about 130 mM NaCl. In contrast, the subfragment 1 that was generated by papain digestion (myosin S1PA), which cleaves RLC 4 kDa away from the NH2-terminal end of the molecule, does not interact with DAN-CaP. Overlay and fluorescent assay in solution showed that CaP binds to isolated RLC, suggesting that the interaction between CaP and subfragment 1 of myosin is due to a direct binding of CaP to RLC. CaP binding to myosin S1SA is stronger than to subfragment 2 in physiological salt concentrations. CaP binding to myosin head strengthened upon phosphorylation of RLC by Ca2+/calmodulin-dependent myosin light chain kinase. We suggest that CaP binds to subfragment 1 of myosin, exclusively via the NH2-terminal end of RLC, and this interaction could play a role in regulation of the actin-myosin interaction in smooth muscle contractility.
Szymanski
P T
PT
Center for Swallowing and Motility Disorders, VA Medical Center, West Roxbury, Massachusetts 02132, USA.
Goyal
R K
RK
D000199:Actins / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D008840:Microfilament Proteins; D008970:Molecular Weight; D009119:Muscle Contraction / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D018994:Myosin Light Chains; D010446:Peptide Fragments; D000096985:Calponins