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 cardiomyopathies that interfere with normal muscle contraction. 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 dynamics 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 immediately in conventional MD simulations suggesting that only hydrophobic patch binders stabilized the open conformation. Additionally, differences in the free energy of hydrophobic patch opening of hypertrophic (HCM) and dilated cardiomyopathy (DCM) cTnC systems were investigated. Molecular dynamics and umbrella sampling simulations revealed a lower free energy of opening for the HCM mutations A8V and A31S, as well as the calcium-sensitizing mutation L48Q. The DCM mutations, Y5H, Q50R, and E59D/D75Y, all exhibited a higher free energy of opening. In conclusion, our developed simulation protocol presents a novel approach to study calcium sensitivity modulation by small molecules and mutations and facilitates a molecular understanding of cardiac muscle contraction.

Mechanism of calcium sensitivity modulation of cardiac troponin C by small molecules illuminated by umbrella sampling simulations

ACS Fall 2021

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