Mechanical Stability of the Cardiac Myosin Binding Protein-C (CMybP-C) Motif is Phosphorylation-Dependent

Abstract

Enhanced cardiac contractility results from phosphorylation within the cMyBP-C motif domain by affecting the interaction of cMyBP-C's N-terminus with actin and/or myosin. We investigated whether motif (i.e. C1C2 linker) phosphorylation alters its structural conformation by characterizing the force-extension behavior of expressed N-terminal cMyBP-C fragments (i.e.C0C3) that contain the motif using atomic force spectroscopy. Proteins were adsorbed onto glass slides, bound non-specifically to SiNi AFM probes, and stretched at 700nm/s. Force-extension traces showed the characteristic sawtooth shape of unfolding immunoglobulin domains, and were well fitted by the wormlike chain model. The model fits produced a median persistence length of 0.36nm and an average C0C3 contour length of 71.5nm, suggesting that the motif is disordered and freely extensible when unphosphorylated. PKA phosphorylation did not change immunoglobulin unfolding force or persistence length but decreased the contour length by 27nm, suggesting that the motif assumed a folded configuration that is stable under load. This was investigated further by varying the extent of phosphorylation using phosphomimetic mutants, in which serines 273, 282, and 302 were replaced with different combinations of alanines or aspartic acids in order to mimic the unphosphorylated and phosphorylated states respectively. While substitution with all alanines or all aspartic acids produced similar results to the control and PKA treated fragments respectively, phosphorylation at only one or two sites did not reduce contour length significantly. This implies that phosphorylation at all three sites is required for motif folding to be stable under load, while partial phosphorylation may result in a fold which is readily extended at low force. In conclusion, cMyBP-C phosphorylation could modulate the cMyBP-C's N-terminal domain interactions with actin and/or myosin by folding the motif in such a way that binding sites for actin and/or myosin are hidden.