Competitive Binding to Myosin S2 by C-Protein, Anti-S2 Peptides, and Antibodies Modulate Myosin Coiled Coil Stability and Myofibril Contractility

Abstract

The myosin subfragment 2 (S2) provides a flexible linkage of myosin heads to the thick filament that is modulated by S2 binding proteins in single molecule assays and myofibrillar contractility. Myosin binding protein C (C-protein), S2 targeted antibody and anti-S2 peptide (modified poly-l-lysine residues) induced antagonistic effects on S2 stability. Competitive ELISA (c-ELISA), myofibrillar contractility assays, and gravitational force spectroscopy (GFS) detected competitive binding between the antibody and anti-S2 peptide for binding S2. The c-ELISA results indicated that the anti-S2 peptide out-competes the antibody in binding to both human cardiac myosin S2 and, to a lesser degree, rabbit skeletal myosin S2. The GFS confirmed competition between the antibody and the anti-S2 peptide by reducing the number of beads attached to myosin via the antibody when the myosin was pre-incubated with anti-peptide. The few GFS measurements observed were consistent with control myosin that did not have anti-S2 peptide bound. In contrast, GFS performed with myosin pre-incubated with C-protein indicated that binding of C-protein to myosin filament stabilized the myosin coiled coil compared to controls without C-protein. Thus C-protein stabilized S2 by binding to a different site on myosin than the anti-S2 peptide. Myofibrillar contractility assay results depicted that binding of antibody to myosin S2 resulted in destabilizing the myosin coiled coil while binding anti-S2 peptide stabilized the coiled coil even in the presence of the antibody. Destabilizing the coiled coil increased contractility observed in myofibrils incubated with antibody compared to untreated myofibrils while stabilizing the coiled coil with the anti-S2 peptide decreased myofibrillar contractility. The results suggest that the myosin S2 performs a sensitive function in muscle contraction that can be modulated by proteins that bind to it.