The effect of Mg²⁺ on Ca²⁺ binding to cardiac troponin C in hypertrophic cardiomyopathy associated TNNC1 variants

Cardiac troponin C (cTnC) is the critical Ca²⁺ -sensing component of the troponin complex. Binding of Ca²⁺ to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca²⁺ binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca²⁺ binding to site II of cTnC. However, work by several groups including ours suggest that Mg²⁺ , which is several orders of magnitude more abundant in the cell than Ca²⁺ , may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg²⁺ may decrease site II Ca²⁺ -binding in both N-terminal and full-length cTnC. Here, we explore the binding of Ca²⁺ and Mg²⁺ to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca²⁺ and Mg²⁺ . Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg²⁺ in modifying the Ca²⁺ affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg²⁺ both at baseline and when elevated on modifying the Ca²⁺ binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction.