Mechanism of Cardiac Troponin C Calcium Sensitivity Modulation by Small Molecules Illuminated by Umbrella Sampling Simulations

Cardiac troponin C (cTnC) binds intracellular calcium and subsequently cardiac troponin I (cTnI), initiating cardiac muscle contraction. Due to its role in contraction, cTnC has been a therapeutic target in the search for small molecules to treat conditions that interfere with normal muscle contraction like the heritable cardiomyopathies. Structural studies have shown the binding location of small molecules such as bepridil, dfbp-o, 3-methyldiphenylamine (DPA), and W7 to be a hydrophobic pocket in the regulatory domain of cTnC (cNTnC) but have not shown the influence of these small molecules on the energetics of opening this domain. Here we describe an application of an umbrella sampling method used to elucidate the impact these calcium sensitivity modulators have on the free energy of cNTnC hydrophobic patch opening. We found that all these molecules lowered the free energy of opening in the absence of the cTnI, with bepridil facilitating the least endergonic transformation. In the presence of cTnI, however, we saw a stabilization of the open configuration due to DPA and dfbp-o binding, and a destabilization of the open configuration imparted by bepridil and W7. Predicted poor binding molecule NSC34337 left the hydrophobic patch in under 3 ns in conventional MD simulations suggesting that only hydrophobic patch binders stabilized the open conformation. In conclusion, this study presents a novel approach to study the impact of small molecules on hydrophobic patch opening through umbrella sampling, and it proposes mechanisms for calcium sensitivity modulation.