Functional importance of Ca 2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Abstract

Ca 2+-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca 2+ due to substitutions of the amino acid residues responsible for Ca 2+ liganding. To evaluate the functional importance of the Ca 2+-deficient N-terminal lobe in the Ca 2+-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, TnC RA, and TnC SA, respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC AA). According to equilibrium dialysis using 45Ca 2+, TnC RA, and TnC SA bound stoichiometrically 3 mol Ca 2+/mol and 1 mol Ca 2+/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280–290 nm upon Ca 2+ binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6 M urea, if Ca 2+ was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca 2+-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC SA conferred as high a Ca 2+ sensitivity to Mg-ATPase activity of rabbit actomyosin–akazara scallop tropomyosin as did the troponin containing TnC AA, whereas the troponin containing TnC RA conferred virtually no Ca 2+ sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca 2+.