Cross-Linking between the Regulatory Regions of Troponin-I and Troponin-C Abolishes the Inhibitory Function of Troponin.

摘要

We reported previously that both residues 48 and 82 on opposite sides of troponin-C's (TnC's) N-terminal regulatory hydrophobic cleft photo-cross-linked to Met121 of troponin-I (TnI) [Luo, Y., Leszyk, J., Qian, Y., Gergely, J., and Tao, T. (1999) Biochemistry 38, 6678-6688]. Here we report that the Ca(2+)-absent inhibitory activity of troponin (Tn) was progressively lost as the extent of photo-cross-linking increased. To extend these studies, we constructed a mutant TnI with a single cysteine at residue 121 (TnI121). In Tn complexes containing TnI121 and mutant TnCs with a single cysteine at positions 12, 48, 82, 98, or 125 (TnC12, TnC48 etc.), TnI121 formed disulfide cross-links primarily with TnC48 and TnC82 when Ca(2+) was present, and with only TnC48 when Ca(2+) was absent. These results indicate that TnI Met121 is situated within the N-domain hydrophobic cleft of TnC in the presence of Ca(2+), and that it moves out of the cleft upon Ca(2+) removal but remains within the vicinity of TnC. Activity assays revealed that the Met121 to Cys mutation in TnI121 reduced the Ca(2+)-present activation of Tn, indicating that Met121 is important in hydrophobic interactions between this TnI region and TnC's N-domain cleft. The formation of a disulfide cross-link between TnI121 and TnC48 or TnC82 abolished the Ca(2+)-absent inhibitory activity of Tn, indicating that the movement of the Met121 region of TnI out of TnC's N-domain cleft is essential for the occurrence of further events in the inhibitory process of skeletal muscle contraction. On the basis of these and other results, a simple mechanism for Ca(2+) regulation of skeletal muscle contraction is presented and discussed.