Non-Equilibrium Molecular Dynamics to Simulate Shear Stress on Angiotensin II Type 1 (AT1) Receptor

Abstract

Cardiac muscle contraction is regulated by the calcium-binding component of the Troponin Complex, cardiac Troponin C (cTnC), in response to an increase in cytosolic calcium levels. Interaction with calcium is followed by a conformational change, which alters the interactions between troponin complex proteins and mediates the interaction between the thin and thick filaments. Mutations that affect the ability of cTnC to bind calcium are hypothesized to increase or decrease calcium affinity, and thereby predicted to induce hypertrophic or dilated cardiomyopathies, respectively. Cardiomyopathy-associated mutations in the N-Domain of cTnC were selected for this study. These mutations include A8V, L29Q, A31S, L48Q, and C84Y. Equilibrium molecular dynamics simulations were used to model the structural effects of these mutations, and their functional impact assessed using Potential of Mean Force (PMF) calculations, and Isothermal Titration Calorimetry (ITC). Very little structural variation was observed in the mutant protein models. Despite this, large differences in the free energy of calcium binding were observed as a function of these sequence substitutions. The results from ITC are, in some cases, at odds with those derived from MD simulations. These differences may be due to the effect of the mutations on the energetics of the transition between the open and closed conformation of the N-cTnC molecule that are not accessible to the timescales of the MD simulations, but will be readily measured by ITC. Taken together, these results demonstrate that mutations associated with Familial Hypertrophic Cardiomyopathy can act through the alterations in the thermodynamic properties of calcium binding and conformational changes.