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

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.

Umbrella Sampling Simulations Measure Switch Peptide Binding and Hydrophobic Patch Opening Free Energies in Cardiac Troponin

“…Computational methods can facilitate the study of these processes . Previous efforts have been able to utilize molecular dynamics (MD) and free energy methods to study Ca 2+ binding to cTnC site II. − MD methods have also been able to investigate disruptions in dynamics upon the introduction of mutations that alter site II’s Ca 2+ sensitivity or mutations that have been linked with disease. − Enhanced sampling methods, umbrella sampling (US), and long timescale simulations have been utilized to study the energetic pathway for the transition between Ca 2+ -unbound and Ca 2+ -bound forms. − Simulations have also incorporated cTnI into the system to elucidate how other subunits in the cTn complex can affect the dynamics of cTnC. − Computer-aided drug discovery has also proven effective in helping identify potential therapeutics for heart disease by targeting the cTn complex. − …”

“…Our lab previously explored this hypothesis by determining the volume sampled by the cTnI SP during MD simulations. We saw no significant difference in cTnI SP effective concentration between wild type (WT) and mutations in the cTnI inhibitory peptide (residues 137–148), leading us to search for other computational methods to explore the effects of mutations on cTnC dynamics.…”

Umbrella Sampling Simulations Simultaneously Measure Switch Peptide Binding and Hydrophobic Patch Opening Free Energies in Cardiac Troponin

“…Our lab previously explored this hypothesis by determining the volume sampled by the cTnISP during MD simulations. We saw no significant difference in cTnISP effective concentration between WT and mutations in the cTnI inhibitory peptide (residues 137-148), 19 leading us to search for other computational methods to explore the effects of mutations on cTnC dynamics.…”

“…[15][16][17][18] Simulations have also incorporated cTnI into the system to elucidate how other subunits in the cTn complex can affect the dynamics of cTnC. [19][20][21][22][23][24] Computer aided drug discovery has also proven effective in helping identify potential therapeutics for heart disease by targeting the cTn complex. [25][26][27][28][29] How mutations located in regions not involved in either the coordination of Ca 2+ to site II or the cTnISP-cTnCHP binding event can alter the overall dynamics of muscle contraction, remains underexplored.…”

Computational Exploration and Characterization of Potential Calcium Sensitizing Mutations in Cardiac Troponin C

“…We successfully predicted the correct calcium binding affinity trends of those mutations compared to the wild-type. Much work has also been done to study the dynamics and energetics of the hydrophobic patch opening and the potential effect of mutations in this region. − Additionally, the dynamics and effects of mutations in other parts of the cTn complex, particularly cTnI, have been explored. − Finally, thermodynamic integration has been previously applied to study calcium and magnesium binding to site II. , In this work, we rationally explore several presumed Ca 2+ sensitizing mutations based on protein stability point mutation predictions and characterize their effects with ASMD and TI. We hope that this publication will be able to serve as a guide for potential future protein design studies.…”