Impact of anti-S2 Peptides on myosin S2 Isoforms and HCM Mutants

Abstract

Myosin subfragment-2 (S2) is an unstable coiled coil that influences the binding of myosin heads to actin. Instability in the myosin S2 could underlie one of the causes for hyper-contractility in Hypertrophic Cardiomyopathy (HCM). Supporting this hypothesis, HCM mutations E924K and E930del in myosin S2 reduced myosin S2 coiled coil stability compared to wild type as measured by Forester's Resonance Energy Transfer (FRET) and Gravitational Force Spectroscopy (GFS). To modulate the stability of the S2, anti-S2 peptides called stabilizer and destabilizer peptides because they are designed to increase and decrease the stability of the MYH7 S2 coiled coil, were tested on a range of human S2 peptide sequences. The anti-S2 peptides were tested on myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by stabilizer and decreased by destabilizer across the cardiac and skeletal myosin S2 peptides but not for smooth muscle isoform. Dose-response curves for FRET assays indicated that both peptides bound at nanomolar concentrations to wild type cardiac and skeletal, but not smooth muscle isoforms. The wild type cardiac S2 peptide required more force to unzip than skeletal, smooth, or mutant peptides in the absence of stabilizer peptide. Effectively, the stabilizer peptide improved the stability of both HCM mutants (E924K and E930del). The stabilizer peptide enhanced dimer formation in mutant cardiac, and skeletal myosin S2 peptides, and destabilizer increased flexibility of cardiac and skeletal myosin S2 wild type peptide. This study demonstrates that the structural stability of myosin S2 can be altered by designed small peptides as well as mutations. These anti-S2 peptides share similar specificities and high affinities for the tested striated muscle isoforms but not the smooth muscle myosin isoform.