Computational Methods Elucidate Consequences of Mutations and Post-translational Modifications on Troponin I Effective Concentration to Troponin C.

Ca2+ binding to cardiac troponin C (cTnC) causes a conformational shift that exposes a hydrophobic patch (cTnCHP) for binding of the cTnI switch peptide (cTnISP), ultimately resulting in contraction of the heart. The inhibitory peptide (cTnIIP), attached at the N-terminal end of the cTnISP, serves as a tether for the cTnISP to the rest of the troponin complex. Due to this tethered nature, the cTnISP remains within proximity of the hydrophobic patch region, resulting in the cTnCHP experiencing an elevated "effective concentration" of the cTnISP. Mutations to the cTnIIP region have been hypothesized to cause disease by affecting the ability of the cTnISP to "find" the hydrophobic patch, resulting in alterations to the heart's ability to contract normally. We tested this hypothesis using molecular dynamics (MD) simulations of the troponin complex using a model that contained all three subunits of troponin: C, I, and T. We developed methods that allowed us to quantitatively measure the effective concentration of the cTnISP from the simulations. A significant reduction in the cTnISP effective concentration was observed when the cTnIIP was removed from the system, showcasing the importance of a tethered cTnISP. Through accelerated MD methods, we proposed the minimum effective concentration of a tethered cTnISP to be approximately 21 mM. Modification of the cTnIIP via PKC-mediated phosphorylation of T143 was shown to significantly increase the estimated effective concentration of cTnISP, help the cTnISP find the cTnCHP more effectively, and maintain the relative shape of the cTnIIP when compared to the native model. All of these data indicate that pT143 may be able to help promote binding of cTnISP to the cTnCHP. We then tested six mutations within the cTnIIP region that are known cTnC Ca2+-sensitizing mutations and have been linked with cardiomyopathy. We did not observe a significant reduction in the effective concentration upon the introduction of these mutations; however, we did observe increased variability in the flexibility and dynamics of the cTnIIP region when compared to native. Our observations led us to hypothesize that the mechanism by which these cardiomyopathic mutations affect Ca2+ sensitivity is by altering the off rate of cTnISP from the hydrophobic patch.