FHC-Linked Myosin Regulatory Light Chain Mutations A13T and K104E Do Not Generate Major Structural Changes Sufficient to Affect Binding of Myosin to Actin

Abstract

A sarcomere, the essential unit of cardiac muscle, consists of myosin, actin, troponin and tropomyosin. The interaction among these proteins generates muscle contraction. The myosin Regulatory Light Chain (RLC) attaches to the myosin heavy chain (MHC) IQ motif and structurally supports the lever arm domain, known to propagate minute conformational changes between the actin and myosin filaments. Mutations in the RLC may therefore cause alterations in the lever arm structure, thus leading to cardiac dysfunction. Here, we investigated two Familial Hypertrophic Cardiomyopathy (FHC) mutations in RLC, A13T (alanine to threonine) and K104E (lysine to glutamate) and hypothesized that these RLC mutants may lead to cardiomyopathy by changing the lever arm structure, the interaction with the MHC and ultimately impairment of the binding of myosin to actin. To test our hypothesis, I performed the following experiments: 1) Titration of the RLC-depleted porcine myosin with different concentrations of recombinant human cardiac RLC wild-type (WT) and A13T and K104E mutants to study the RLC-MHC binding properties (affinity, kinetics); 2) Study of the actin-myosin interaction by fluorescence and light scattering measurements. My preliminary data shows that the WT-RLC displays a clearly cooperative binding to the MHC, however, a reduction in cooperativity was observed in both mutants. In parallel, the binding affinity of A13T and K104E was slightly decreased (1.3 fold and 1.4-fold increase in Kd, respectively). Fluorescence and light scattering measurements monitoring the binding of WT or mutant-myosin to pyrene-labeled actin demonstrated a very strong binding with no mutant dependent changes in Kd. These results suggest that any structural changes that may be caused by these two FHC-RLC mutations are not sufficient to affect the myosin-actin binding. Supported by NIH-HL071778 (DSC).