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

Abstract

Ca²⁺ 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 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 cTnI_IP 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 cTnI_SP from the hydrophobic patch.