Abstract
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.