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

Ca 2+ binding to cardiac troponin C (cTnC) causes a conformational shift that exposes a hydrophobic patch (cTnC HP ) for binding of the cTnI switch peptide (cTnI SP ), ultimately resulting in contraction of the heart. The inhibitory peptide (cTnI IP ), attached at the N-terminal end of the cTnI SP , serves as a tether for the cTnI SP to the rest of the troponin complex. Due to this tethered nature, the cTnI SP remains within proximity of the hydrophobic patch region, resulting in the cTnC HP experiencing an elevated "effective concentration" of the cTnI SP . Mutations to the cTnI IP region have been hypothesized to cause disease by affecting the ability of the cTnI SP 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 cTnI SP from the simulations. A significant reduction in the cTnI SP effective concentration was observed when the cTnI IP was removed from the system, showcasing the importance of a tethered cTnI SP . Through accelerated MD methods, we proposed the minimum effective concentration of a tethered cTnI SP to be approximately 21 mM. Modification of the cTnI IP via PKC-mediated phosphorylation of T143 was shown to significantly increase the estimated effective concentration of cTnI SP , help the cTnI SP find the cTnC HP more effectively, and maintain the relative shape of the cTnI IP when compared to the native model. All of these data indicate that pT143 may be able to help promote binding of cTnI SP to the cTnC HP . We then tested six mutations within the cTnI IP region that are known cTnC Ca 2+ -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 cTnI IP region when compared to native. Our observations led us to hypothesize that the mechanism by which these cardiomyopathic mutations affect Ca 2+ sensitivity is by altering the off rate of cTnI SP from the hydrophobic patch.