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

Calcium-dependent heart muscle contraction
is regulated by the
cardiac troponin protein complex (cTn) and specifically by the N-terminal
domain of its calcium binding subunit (cNTnC). cNTnC contains one
calcium binding site (site II), and altered calcium binding in this
site has been studied for decades. It has been previously shown that
cNTnC mutants, which increase calcium sensitization may have therapeutic
benefits, such as restoring cardiac muscle contractility and functionality
post-myocardial infarction events. Here, we computationally characterized
eight mutations for their potential effects on calcium binding affinity
in site II of cNTnC. We utilized two distinct methods to estimate
calcium binding: adaptive steered molecular dynamics (ASMD) and thermodynamic
integration (TI). We observed a sensitizing trend for all mutations
based on the employed ASMD methodology. The TI results showed excellent
agreement with experimentally known calcium binding affinities in
wild-type cNTnC. Based on the TI results, five mutants were predicted
to increase calcium sensitivity in site II. This study presents an
interesting comparison of the two computational methods, which have
both been shown to be valuable tools in characterizing the impacts
of calcium sensitivity in mutant cNTnC systems.