Activation of Cardiac Thin Filaments by N-Terminal Domains of Cardiac Myosin Binding Protein C

Abstract

We have used double mixing stopped-flow fluorescence to study the effect of the soluble N-terminal C1C2 domain of cMyBP-C on the activation of product dissociation from myosin-S1-mdADP-Pi by native porcine thin filaments. Product dissociation from cardiac actomyosin is an ordered process in which phosphate dissociation is rate limiting (AM-mdADP-Pi → AM-mdADP → AM) and can therefore be measured by the rate of mdADP dissociation. Myosin-S1 was mixed with an equal concentration of the fluorescent ATP analogue, mdATP, and held in a delay line 2 seconds for the nucleotide to bind and be hydrolyzed (M + mdATP → M-mdATP ↔M-mdADP-Pi). The mixture was subsequently mixed with native cardiac thin filaments, 1 mM MgATP, 1 mM EGTA and expressed mouse cMyBP-C domains C1C2. In control experiments cMyBP-C was either replaced with NEM-S1 (a catalytically inactive form of myosin-S1, modifed with N-ethylmaleimide, which binds to actin in the presence of ATP), rigor myosin-S1 (in the absence of ATP) or 0.1 mM CaCl2. Maximal activation of the rate of product dissociation by cMyBP-C domains C1C2 is the same as observed with saturating rigor-myosin-S1 or 0.1 mM calcium and greater than observed with NEM-S1. These experiments were done at physiological ionic strength (0.18 M KAc, 10 mM Mops, 2 mM MgCl2, pH 7) to eliminate non specific ionic interaction that might occur at lower ionic strength. Cardiac cMyBP-C domains C1C2 not only bind to actin at a similar position to rigor myosin-S1 but substoichiometric concentrations (1 cMyBP-C C1C2 per 7 actin subunits) are as effective as rigor myosin-S1 at activating cardiac thin filament in the absence of calcium.