Myosin binding protein-C (MyBP-C) is a key sarcomeric protein that regulates muscle contractility through interactions between its N-terminus and actin and myosin. Of the three paralogs, slow skeletal (sMyBP-C) and cardiac (cMyBP-C) are phosphorylated by protein kinase A (PKA) in response to β-adrenergic signaling to enhance muscle contractility. Although both are phosphorylated in their N-terminal regions, the patterns differ: human sMyBP-C at one site within the proline/alanine-rich linker (PAL) preceding the C1 domain, and cMyBP-C at four sites within the M-domain located between domains C1 and C2. Despite the importance of phosphorylation, how it changes the structural dynamics of the N-terminal regions to alter actin and myosin interactions remains unclear. To address this, we examined phosphorylation effects on the structural dynamics of sMyBP-C and cMyBP-C N-terminal fragments (sC1-C2 and cC0-C2) using time-resolved fluorescence resonance energy transfer (TR-FRET). Phosphorylation of sC1-C2 shows small effects on the structural dynamics of the disordered PAL but substantial alterations in the dynamics between specific sites in the structured C1 and C2 domains with increases of 0.5 ± 0.1 nm in the center distance and 0.8 ± 0.1 nm in the full-width at half maximum. In contrast, phosphorylation of cC0-C2 did not affect distances and disorder between the analogous sC1-C2 sites. A sMyBP-C splice variant excluding 25 amino acids and the PKA recognition site in the PAL affects actin motility differently than the sMyBP-C variant including these 25 amino acids. Comparison of FRET results between these isoforms of sC1-C2 reveals additional mechanistic insight into sMyBP-C regulation. This work supports the idea that phosphorylation and splicing uniquely affect sMyBP-C N-terminal conformation and dynamics to fine-tune skeletal muscle performance, distinct from phosphorylation effects on cMyBP-C N-terminal structure and dynamics in regulation of cardiac muscle contractility.
Seffrood et al. (Sun,) studied this question.