Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites

Abstract

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca²⁺ binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin–tropomyosin–actin complex. Herein, we report a calorimetric study of Ca²⁺ binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca²⁺ binding to both the high-affinity and the low-affinity domain of HcTnC. Ca²⁺ binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca²⁺ binding to the regulatory domain. This pattern stems from changes that occur at the Ca²⁺ site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC_1–89 support this claim. The values of the Gibbs free energy for Ca²⁺ binding to the N-domain in the full-length protein and to the isolated domain (HcTnC_1–89) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (∆C _p ) from a plot of ∆H verses temperature and may provide evidence for positive cooperativity of Ca²⁺ binding to the C-domain.