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.

Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: Potential role for coiled coil interaction

“…Recently, Jha et al (6) showed that the deletion of 57 carboxyterminal residues of TnT eliminates TnI binding. Our results showing the involvement of HR domains of TnT and TnI in binary interaction are consistent with the above-mentioned reports.…”

“…However, the structural basis for its role in this process is not clear. Recent mutagenesis studies have identified the region of fast skeletal TnT that contributes to the Ca 2ϩ sensitivity of the thin filament-based regulatory system (6).…”

“…The available studies in the literature using either their proteolytic fragments that are soluble, or intact proteins at high salt concentrations, have implicated various regions of TnT, spanning residues 71-258, which interacted with TnI (7)(8)(9)(10). Recent studies on the interaction of in vitro expressed deletion mutants of TnI and TnT by HPLC suggested that residues 1-120 of TnI bind to residues 202-258 of TnT (6). Clearly, the precise interaction sites of TnI and TnT remain to be examined for a better understanding of the vertebrate striated muscle contraction process.…”

Tissue-specific interactions of TNI isoforms with other TN subunits and tropomyosins in C. elegans: The role of the C- and N-terminal extensions

“…Immunoblotting, cross-linking and competitive labeling studies on the rabbit fast skeletal TNI and TNT suggest that the TNI 58-107 segment bound to TNT [22][23][24]. The atomic structure of the human cardiac troponin core complex shows that the helical part of H2 (I) interacted to H2 (T2) [4].…”

Developmentally Regulated, Alternative RNA Splicing-generated Pectoral Muscle-specific Troponin T Isoforms and Role of the NH2-terminal Hypervariable Region in the Tolerance to Acidosis

“…Studies of TnT structure-function relationships have revealed that the COOH-terminal T2 fragment of TnT binds to the central region of Tm, whereas the central region of the TnT polypeptide has been shown to interact with the head-to-tail overlapping region of Tm (13)(14)(15)(16). The COOHterminal T2 region of TnT also anchors the TnI-TnC binary complex formation in a direct, Ca 2ϩ -independent manner (17,18). Resonance energy transfer experiments showed that TnT, but not TnC, caused an elongation of the TnI molecule (19).…”