IDENTIFICATION AND MUTAGENESIS OF A HIGHLY CONSERVED DOMAIN IN TROPONIN-T RESPONSIBLE FOR TROPONIN-I BINDING - POTENTIAL ROLE FOR COILED-COIL INTERACTION

Troponin T (TnT), a thin filament myofibrillar protein, is essential f or the Ca2+ regulation of striated muscle contraction in vertebrates, both in vivo and in vitro. To understand the role of TnT in this proce ss, its interaction with two other troponin components, troponin I (Tn I) and tropo nin C (TnC) was examined by using the yeast two hybrid sy stem, which is a genetic approach to detect protein-protein interactio ns, Computer assisted analysis of phylogenetically distant TnT amino a cid sequences unveiled a highly conserved protein domain that is chara cterized by a heptad repeat (HR) motif with a potential for alpha-heli cal coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TnI sequences, These protein mot ifs appeared to be the regions where TnI-TnT interaction may take plac e, Deletions and point mutations in TnT, which disrupted its HR motif, severely reduced or abolished TnI binding, but binding to TnC was not affected, indicating that the TnT-TnI and TnT-TnC binary interactions can be uncoupled. Remarkably, the truncated fragments of TnT and TnI in which the HR motifs were retained showed binary interaction in the yeast two hybrid system, It was also observed that the formation of th e TnT-TnI heterodimers Is favored over the homodimers TnT-TnT and TnI- TnI. These results indicate that the evolutionarily conserved HR motif s may play a role in TnT-TnI dimerization, presumably through the form ation of alpha-helical coiled coils.