Calcium-dependent regulation of smooth muscle calponin by S100.

Calponin, a major calmodulin-, actin-, and tropomyosin-binding protein in smooth muscle, interacted with brain S100 and the properties of the interaction were investigated in detail. From fluorescence labeling and chemical cross-linking experiments, the apparent Kd value was calculated to be 7 x 10(7) M-1 in the presence of Ca2+ with 1 mol of S100 bound per mol of calponin. The addition of S100 to the mixture of calponin and F-actin caused the removal of calponin from actin filaments in the presence of Ca2+ but not in the presence of EGTA or Zn2+. Ca2+ and S100 could relieve calponin-induced actomyosin Mg(2+)-ATPase inhibition. Both the removal of calponin from F-actin and the restoration of ATPase inhibition by S100 were more effective than those by calmodulin. At low ionic strength, the binding was observed irrespective of Ca2+ concentration and it was greatly weakened with increasing salt concentration. The formation of the complex in the presence of Ca2+ was less sensitive, with only 45% inhibition at 100 mM NaCl, where the complex in the absence of Ca2+ had almost disappeared. This was confirmed by S-100 Sepharose 4B chromatography. Addition of Ca2+ and S100 also led to a decrease in the affinity of calponin for tropomyosin. Domain mapping with chymotryptic digestion revealed that the S100 binding site resided within the N-terminal 22 kDa fragment of calponin, where the bindings of calmodulin and actin also occur.