(E-162) An Investigation of the Molecular Dynamics of the ß-MHC R369Q Mutation

Mutations in β-myosin heavy chain (MHC) have been implicated in the manifestation of cardiomyopathies. One such disease, dilated cardiomyopathy (DCM), results in a progressive remodeling of the heart, causing a dilation of the chambers that makes it more difficult for the heart to pump blood. This can lead to progressive heart failure. Current methods of treating cardiomyopathies are ineffective and target secondary manifestations of heart disease rather than the mutagenic etiology. Our aim is to better understand the pathogenesis of cardiomyopathies by studying a particular disease-associated mutation, R369Q, that resides on loop 4 of the β-MHC structure. Though crystal structures are useful tools in discerning structure, they are a static representation of an inherently dynamic protein. We alternatively leverage molecular dynamics methods because they allow for a detailed atomic-scale analysis of a dynamic simulation of the β-MHC structure.

Molecular dynamics were performed in triplicate for both the wild type and R369Q structure in the pre-powerstroke M.ADP.Pi state (PDB: 5N6A) using AMBER20. Each of these six simulates were run for 500 ns. The simulations were then processed in CPPTRAJ, and calculations and visualizations of root mean squared deviation and fluctuation (RMSD and RMSF) were done using Python. Particularly, we employed these methods to analyze the differences in structural stability and nucleotide-binding pocket coordination in the mutant versus the wild type.

Measurements of RMSD for each of the six simulations demonstrate that the R369Q simulations have a higher RMSD on average than the wild type. This indicates that the R369Q structure is sampling more conformations over time than the wild type, which could imply increased instability of the mutant structure. This is further supported by RMSF data that show the average flexibility for the majority of residues in the R369Q mutation is greater than that of the corresponding wild type residues. Together, these two analyses suggest that the overall structure of the R369Q β-MHC is inherently less structurally stable than the wild type, leading to increased flexibility and conformation sampling as the protein equilibrates its structure. Additionally, we examined contacts within Switch I, a loop adjacent to the nucleotide-binding pocket of myosin that contributes to structural communication between the actin-binding cleft, where the mutation is located, and the nucleotide-binding pocket. Here, we observed a significant loss of several contacts between Switch I and the Upper 50 kDa Domain in the R369Q structure. This loss of contacts could impair structural signal transduction between the actin-binding cleft and the nucleotide-binding pocket, altering the ATPase cycle of myosin and affecting contractility. From the results of these three analyses, we postulate that the R369Q mutation introduces structural instability into the myosin structure and disrupts the inter-protein communication of structural changes that drives the ATPase cycle. These findings provide a strong hypothesis for some of the reasons R369Q mutation impairs contraction and is associated with DCM.