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.

Omecamtiv mecarbil (OM) is a positive inotrope that is thought to bind directly to an allosteric site of the β-cardiac myosin. The drug is under investigation for the treatment of systolic heart failure. The drug is classified as a cardiac myosin modulator and has been observed to affect multiple vital steps of the cross-bridge cycle including the recovery stroke and the chemical step. We explored the free-energy surface of the recovery stroke of the human cardiac β-myosin in the presence of OM to determine its influence on this process. We also investigated the effects of OM on the recovery stroke in the presence of genetic cardiomyopathic mutations R712L, F764L, and P710R using metadynamics. We also utilized the method of transition path sampling to generate an unbiased ensemble of reactive trajectories for the ATP hydrolysis step in the presence of OM that were able to provide insight into the differences observed due to OM in the dynamics and mechanism of the decomposition of ATP to ADP and HPO4 2–, a central part of the power generation in cardiac muscle. We studied chemistry in the presence of the same three mutations to further elucidate the effect of OM, and its use in the treatment of cardiac disease.

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Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disease which affects $1 in 200 individuals. HCM results from mutations in sarcomeric proteins, but an understanding of how these mutations affect contractility at the molecular level is lacking. Here, we focus on the two proteins that together house $70% of the known HCM mutations: b-cardiac myosin and cardiac myosin binding protein-C, cMyBPC (a regulatory protein in the myosin thick filament). Functionally, cardiac myosin in the sarcomere can exist in an 'open' state where the heads are available for interaction with actin, and a 'closed' state where myosin heads fold back onto their own tails and don't bind actin. An increase in the proportion of myosin molecules in the 'open' state (N a ) can cause an increase in the power generation of the sarcomere. We have previously shown that several HCM mutations within myosin weaken the 'closed' state of myosin and release active heads, thereby causing hypercontractility by increasing N a . We recently proposed that cMyBPC binds to myosin and sequesters myosin heads in the folded-back 'closed' state, thereby regulating N a . We also proposed that a majority of HCM mutations in cMyBPC affect the ability of cMyBPC to bind and sequester myosin heads. I will present our preliminary biochemical and structural characterization of the binding of wild-type cMyBPC to wild-type b-cardiac myosin, as well as the effects of HCM-causing mutations on that interaction. Using purified recombinant human proteins, we show that cMyBPC binds b-cardiac myosin, resulting in folding back of myosin heads, seen in both functional assays and single-particle electron microscopy images of the complex. We plan to map the interface between the two proteins by using cross-linking mass spectrometry, and parallelly visualize the complex using cryogenic electron microscopy.