Structure of the Mg super(2+)-Loaded C-Lobe of Cardiac Troponin C Bound to the N-Domain of Cardiac Troponin I: Comparison with the Ca super(2+)-Loaded Structure

Cardiac troponin C (cTnC) is the Ca super(2+)-binding component of the troponin complex and, as such, is the Ca super(2+)-dependent switch in muscle contraction. This protein consists of two globular lobes, each containing a pair of EF-hand metal-binding sites, connected by a linker. In the N lobe, Ca super(2+)-binding site I is inactive and Ca super(2+)-binding site II is primarily responsible for initiation of muscle contraction. The C lobe contains Ca super(2+)/Mg super(2+)-binding sites III and IV, which bind Mg super(2+) with lower affinity and play a structural as well as a secondary role in modulating the Ca super(2+) signal. To understand the structural consequences of Ca super(2+)/Mg super(2+) exchange in the C lobe, we have determined the NMR solution structure of the Mg super(2+)-loaded C lobe, cTnC(81-161), in a complex with the N domain of cardiac troponin I, cTnI(33-80), and compared it with a refined Ca super(2+)-loaded structure. The overall tertiary structure of the Mg super(2+)-loaded C lobe is very similar to that of the refined Ca super(2+)-loaded structure as evidenced by the root-mean-square deviation of 0.94 Ae for all backbone atoms. While metal-dependent conformational changes are minimal, substitution of Mg super(2+) for Ca super(2+) is characterized by condensation of the C-terminal portion of the metal-binding loops with monodentate Mg super(2+) ligation by the conserved Glu at position 12 and partial closure of the cTnI hydrophobic binding cleft around site IV. Thus, conformational plasticity in the Ca super(2+)/Mg super(2+)-dependent binding loops may represent a mechanism to modulate C-lobe cTnC interactions with the N domain of cTnI.