Magnesium-calcium exchange in cardiac troponin C bound to cardiac troponin I

Understanding the process of Ca2+/Mg2+ exchange during muscle excitation and relaxation is fundamental to elucidating the mechanism of Ca2+-regulated muscle contraction. During the resting phase, the C-domain of cardiac troponin C may be occupied by either Ca2+ or Mg2+. Here, complexes of recombinant cardiac troponin C(81-161) and the N terminus of cardiac troponin I, representing residues 33-80, were generated in the presence of saturating Mg2+. Heteronuclear multi-dimensional nuclear magnetic resonance experiments were used to obtain backbone assignments of the Mg2+-loaded complex. In the presence of cardiac troponin I, the affinity of site IV for Mg2+ is increased. Comparison of Mg2+ and Ca2+-loaded complexes reveals that chemical shift differences are primarily localized to metal-binding sites III and IV, defining positions within these sites that have distinct Ca2+/Mg2+ conformations. The observed transition from the Mg2+-loaded to Ca2+-loaded form demonstrates that sites III and IV fill simultaneously with Ca2+ displacing Mg2+. However, even in the absence of excess Ca2+, Mg2+ does not readily displace Ca2+ in the isolated binary complex. Thus, the Mg2+-loaded conformer may only represent a small fraction of the total cardiac troponin C found in the sarcomere. (C) 2000 Academic Press.