Computational Study of the Effect of Point Mutations Perturbing the Recovery Stroke of Human Cardiac Beta-Myosin using Metadynamics

Abstract

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.