Investigation of the Recovery Stroke and ATP Hydrolysis and Changes Caused Due to the Cardiomyopathic Point Mutations in Human Cardiac β Myosin

Abstract

Human cardiac β myosin undergoes the cross-bridge cycle as part of the force-generating mechanism of cardiac muscle. The recovery stroke is considered one of the key steps of the kinetic cycle as it is the conformational rearrangement required to position the active site residues for hydrolysis of ATP and interaction with actin. We explored the free-energy surface of the transition and investigated the effect of the genetic cardiomyopathy causing mutations R453C, I457T, and I467T on this step using metadynamics. This work extends previous studies on Dictyostelium myosin II with engineered mutations. Here, like previously, we generated an unbiased thermodynamic ensemble of reactive trajectories for the chemical step using transition path sampling. Our methodologies were able to predict the changes to the dynamics of the recovery stroke as well as predict the pathway of breakdown of ATP to ADP and HPO4 2– with the stabilization of the metaphosphate intermediate. We also observed clear differences between the Dictyostelium myosin II and human cardiac β myosin for ATP hydrolysis as well as predict the effect of the mutation I467T on the chemical step.

Free-Energy Surfaces of Two Cardiac Thin Filament Conformational Changes during Muscle Contraction

“…The CV has been previously used to simulate a similar conformational shift in both human myosin and dictyostelium myosin II in previous metadynamics studies performed in our group. 20,21 Therefore, a similar approach was deemed appropriate and utilized for the systems in the current study.…”

“…This CV was able to distinctly differentiate between the initial and final states for cTnC, making it an appropriate CV for this simulation. The CV has been previously used to simulate a similar conformational shift in both human myosin and dictyostelium myosin II in previous metadynamics studies performed in our group. , Therefore, a similar approach was deemed appropriate and utilized for the systems in the current study.…”

“…16 Similarly, metadynamics has been applied to study the recovery stroke of two different isoforms of myosin, again supporting the use of metadynamics to uncover the free-energy profile for conformational transitions. 20,21 Here, we simulated the opening of N-cTnC, both saturated and unsaturated, with Ca 2+ to determine the effect that ion binding has on the conformational transition. We also simulated the dissociation of cTnI from actin with N-cTnC in both the open and closed conformations to establish if the conformational state of cTnC had an effect on the energetics of the dissociation.…”

Computational and biophysical determination of pathogenicity of variants of unknown significance in cardiac thin filament

“…These distributions and changes are difficult for an individual to assess, but a more automated process, such as deep machine learning, is exactly tailored to such a task and is a topic of ongoing research. In addition, understanding the pathogenic effects on interactions of the CTF with other proteins in the sarcomere (e.g., the thick filament) is under intensive study ( 49 , 50 ). Moreover, we hope to be able to expand this analysis to other regions of the CTF, such as the C-terminus of cTnT, and to extend the length of simulations in the future.…”

Perspective: Path Sampling Methods Applied to Enzymatic Catalysis

“…The past 3 years has seen a significant expansion in the application of TPS to understanding enzymatic chemistry. We count 9 separate systems from 6 separate groups ,− being studied in this fashion. The technique is clearly gaining in importance, but there remain basic hurdles to more general application.…”