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 I(129−166) complex demonstrated that cardiac troponin I(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 15N transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1−80)/troponin I(129−166) or troponin I(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 I(129−166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin I(129−166) binding the cardiac troponin C/troponin I(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 I(1−80) was used to monitor binding of cardiac troponin I(129−166) to the regulatory domain of cardiac troponin C. The apparent K d for cardiac troponin I(129−166) binding to cardiac troponin C/troponin I(1−80) was 43.3 ± 3.2 μM. After bisphosphorylation of cardiac troponin I(1−80) the apparent K d increased to 59.1 ± 1.3 μM. 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 I(129−166) and provides further evidence for β-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.