Interaction of deletion mutants of troponins I and T: COOH-terminal truncation of troponin T abolishes troponin I binding and reduces Ca2+ sensitivity of the reconstituted regulatory system

Abstract

The interaction between troponin I (TnI) and troponin T (TnT) remains the least understood binary interaction among the regulatory proteins of vertebrate striated muscle. To identify the specific binding domains of TnI and TnT and to evaluate the interactions of TnT with troponin C and tropomyosin (Tm), we generated an NH2-terminal fragment of human fast skeletal beta TnT (TnT1-201; residues 1-201) using site-directed mutagenesis. The mutant protein failed to bind to rabbit skeletal muscle TnI as judged by HPLC, showed reduced TnC binding and reduced ternary troponin (Tn) complex formation, and exhibited a much reduced Ca2+ sensitivity in the reconstituted regulatory system. It is shown that the amount of Tn complex formed by TnT1-201 rather than the activity of the mutant Tn complex affected this Ca2+ sensitivity. Binding of the mutant to Tm was similar to that of intact TnT. These results support the view that the COOH-terminal segment of TnT is necessary for binding to TnI and TnC and Ca2+ sensitivity in the thin filament, whereas its NH2-terminus strongly binds to Tm. To identify the regions of TnI which bind to muscle TnT, we used four recombinant fragments of fast skeletal muscle TnI containing amino acid residues 1-94 (TnI1-94), 1-120 (TnI1-120), 96-181 (TnI96-181), and 122-181 (TnI122-181) and a synthetic peptide, TnI98-114, containing residues 98-114 corresponding to the inhibitory region. Only TnI1-120 showed weak binding to TnT but not to TnT1-201. These results suggest that (i) a region within the NH2-terminal 120 residues of TnI interacts with TnT and (ii) the COOH-terminal residues 202-258 of TnT contain the interaction site of TnI. Overall, our results also imply that residues 159-201 constitute the smallest region of TnT which contributes to the Ca2+ sensitivity of actoS1 ATPase in a reconstituted regulatory system.