Functional role of Ca2+-binding site IV of scallop troponin C

Scallop troponin C (TnC) binds only one Ca2+/mol and the single Ca2+-bindin g site has been suggested to be site IV on the basis of the primary structu re [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-346 8; T. Ojima, Ii. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca2+-binding site I V of akazara scallop (Chlamys nipponensis akazara) TnC in Ca2+-regulation w as investigated using a site-directed mutant with an inactivated site TV (T nC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and w ild-type TnC (TnC(C)). Equilibrium dialysis using Ca-45(2+) demonstrated th at TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca2+/mol, but that TnC-ZEQ and TnC (N) bind virtually no Ca2+. The UV difference spectra of TnC(W) and TnC(C) showed bands at around 280-290 nm due to the perturbation of Tyr and Trp up on Ca2+-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addi tion, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl2, unlike TnC-ZEQ and TnC(N). These results i ndicate that Ca2+ binds only to site IV and that Ca2+-binding causes struct ural changes in both the whole TnC molecule and the C-terminal half molecul e. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N) were shown to f orm soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin i n the presence of scallop tropomyosin was inhibited by scallop TnI and reco vered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the asso ciation with TnI is located in the C-terminal half domain of TnC. Ternary c omplex constructed from scallop TnT, TnI, and Tnc, conferred Ca2+-sensitivi ty to the Mg-ATPase of rabbit actomyosin to the same extent as native tropo nin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca2-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca2+-sensitiv ity. Thus, the major functions of scallop TnC, such as Ca2+-binding and int eraction with TnI, are located in the C-terminal domain, however, the full Ca2+-regulatory function requires the presence of the N-terminal domain.