Abstract
The human hypertrophic cardiomyopathy mutation R453C is associated with a high incidence of sudden cardiac death. We have expressed the motor domain (MD) of human beta-myosin and the human ventricular essential light chain (MLC3) with the R453C mutation using a mouse muscle cell line. We have examined the binding of nucleotides to the MD and actin·MD and compared the kinetic data to our previous study of the wild type MD (Deacon et al 2012). Little change was observed in the binding of ATP or ADP to actin·MD but the two fluorescence changes accompanying ATP binding to MD in the absence of actin were slower. The fast phase, previously assigned to the conformational change accompanying tight binding of ATP to the motor, was slowed by 30%. The slow phase,assigned to the closure of switch 2 and the recovery stroke, associated with the ATP hydrolysis step, were slowed ∼ 3 fold. The R453C mutation is located in a surface loop that links the switch-2 beta strand with the long O-helix that connects to the cardiomyopathy loop in the actin binding site. R453C is therefore in a position to influence both actin binding and switch 2 closure. The surface loop and R453 are conserved only in sarcomeric myosins, suggesting a specific role for the loop in muscle. Supported by NIH grants GM29090 and GM65013 and Wellcome Trust grant 085309.
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