Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca²⁺ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca²⁺ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca²⁺ or Mg²⁺, and two regulatory sites in the N-terminal domain are specific for Ca²⁺. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca²⁺ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca²⁺- and Mg²⁺-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca²⁺. When the regulatory Ca²⁺-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg²⁺, we found significant differences between full-length TnI and Tni(1-159) in their effect on Trp-26. Our results provide the first indication that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca²⁺-dependent interactions with the regulatory domain of TnC.