Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I

Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin 1(129-166) complex demonstrated that cardiac troponin 1(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and N-15 transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1-80)/troponin 1(129-166) or troponin 1(1-167) suggested that troponin I residues 81-128 do not interact strongly with troponin C but likely serve to modulate the interaction of troponin 1(129-166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin 1(129-166) binding the cardiac troponin C/troponin 1(1-80) complex correlate with partial opening of the cardiac troponin C regulatory domain previously demonstrated by distance measurements using fluorescence methodologies. Fluorescence emission from cardiac troponin C(F20W/N51C)(AEDANS) complexed to cardiac troponin 1(1-80) was used to monitor binding of cardiac troponin 1(129-166) to the regulatory domain of cardiac troponin C. The apparent K-d for cardiac troponin 1(129-166) binding to cardiac troponin C/troponin 1(1-80) was 43.3 +/- 3.2 muM. After bisphosphorylation of cardiac troponin 1(1-80) the apparent K-d increased to 59.1 +/- 1.3 muM. Thus, phosphorylation of the cardiac-specific N-terminus of troponin I reduces the apparent binding affinity of the regulatory domain of cardiac troponin C for cardiac troponin 1(129-166) and provides further evidence for beta -adrenergic modulation of troponin Ca2+ sensitivity through a direct interaction between the cardiac-specific amino-terminus of troponin I and the cardiac troponin C regulatory domain.