Investigating mutant hydrophobic patch exposure in troponin C using molecular dynamics-based free energy methods.

Abstract

Contraction of cardiomyocytes is orchestrated through calcium-binding to the N-terminal regulatory domain of cardiac troponin C (NcTnC). This binding event initiates a series of dynamic and conformational changes that expose a hydrophobic patch to which troponin I (TnI) associates. Cardiomyopathies have been linked to mutations in NcTnC that disrupt these precise interactions and modulate free energy of opening. Mutations that lower the free energy of opening have a calcium-sensitizing effect and those that increase free energy of opening have a calcium-desensitizing effect, like mutations implicated with hypertrophic (HCM) and dilated cardiomyopathy (DCM), respectively. Albeit mutations occurring in the loop region of NcTnC have been most extensively studied, information regarding mutations within the hydrophobic patch is lacking. Simulating the closed-to-open transition in NcTnC is challenging and typically requires microsecond timescales just to observe partial opening of the patch. Here, we use targeted molecular dynamics (TMD) to efficiently sample the landscape between the open and closed conformations. Umbrella sampling (US) was employed to predict the free energy of opening by using frames sourced from TMD, and preliminary data corroborates the expected trends for known HCM and DCM mutations in NcTnC. RosettaDesign was used to prepare and score novel mutation candidates by systematically mutating residues at eight different positions in the patch to almost every other amino acid. Based on the ref15_cart scoring function, up to three mutation candidates from each position were selected to proceed to further evaluation with US. In this work, we identify several mutations that are expected to perturb patch opening and present an innovative approach to quantitatively study the influence hydrophobic patch residues play in the molecular basis of myocyte contraction.