Analysis of Free Energy of Opening the Troponin C Binding Pocket for Troponin I using Microsecond Molecular Dynamics Simulations

Abstract

Troponin (Tn), part of the thin filament in cardiomyocytes, plays an important role in calcium signaling events in cardiac muscle contraction. It acts as a Ca2+-dependent switch, activating and deactivating the myofilament leading to contraction and relaxation of the muscle cell. An important mechanism in the regulation of contraction is the opening up of the TnC hydrophobic patch to allow TnI to bind. We performed microsecond molecular dynamics simulations of TnC and two of its mutants (V44Q, E40A) in different states of calcium binding. The dynamics and kinetics of the observed opening transitions allow for the estimation of free energy differences for opening. We obtained values for wildtype Ca2+-bound TnC (∼8 kcal/mol), V44Q Ca2+-bound TnC (3.2 kcal/mol), E40A Ca2+-bound TnC (∼12 kcal/mol) and wildtype apo TnC (∼20 kcal/mol). These results suggest that the mutations have profound impact on the frequency of presenting the hydrophobic patch to TnI. These simulations also corroborate that cardiac wildtype TnC does not open on timescales relevant to contraction without calcium being bound. Additionally, Brownian dynamics simulations are used to investigate TnI association with TnC. Simulations of a full length troponin model elucidate the dynamical interplay between the TnC, TnI and TnT subunits.