Binding of calcium and magnesium to human cardiac troponin C

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca²⁺ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca²⁺-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca²⁺ and Mg²⁺ and a regulatory site (II) that has been thought to bind only Ca²⁺. Binding of Ca²⁺ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca²⁺/Mg²⁺ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca²⁺/Mg²⁺ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg²⁺ caused a 1.4-fold lower affinity for Ca²⁺. These experiments strongly suggest that cytosolic-free Mg²⁺ occupies a significant population of the available site II. Interaction of Mg²⁺ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg²⁺, such as secondary hyperparathyroidism or ischemia, respectively.