Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca2+-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca2+ due to substitutions of the amino acid residues responsible for Ca2+ liganding. To evaluate the functional importance of the Ca2+-deficient N-terminal lobe in the Ca2+-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnCRA, and TnCSA, respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnCAA). According to equilibrium dialysis using 45Ca2+, TnCRA, and TnCSA bound stoichiometrically 3 mol Ca2+/mol and 1 mol Ca2+/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280–290 nm upon Ca2+ binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6 M urea, if Ca2+ was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca2+-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnCSA conferred as high a Ca2+ sensitivity to Mg-ATPase activity of rabbit actomyosin–akazara scallop tropomyosin as did the troponin containing TnCAA, whereas the troponin containing TnCRA conferred virtually no Ca2+ sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca2+.