Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain

Abstract

Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments’ actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20ms) and long (~300ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

“…In addition to the thin filament activating effects of cMyBP-C at low Ca 2+ , cMyBP-C also governs contractility, as evidenced by enhanced unloaded cardiac muscle shortening velocities in cMyBP-C null mice (43). The inhibition of velocity in the presence of cMyBP-C occurs through cMyBP-C's N terminus, as observed previously by ourselves and others (16,19,20,50) and confirmed here (Fig. 6).…”

“…The cMyBP-C domains primarily involved in binding actin appear to be the C1 and M-domains (9,15,16,20), although there is evidence that C0 may be important (10,14). Previous modeling suggested that the C0 and C1 domains could clash with Tm in its blocked position (25,27,28), whereas experiments in which expressed N-terminal fragments were added to skinned cardiac myocytes implicated the ProAla-rich domain between C0 and C1 in modulating Ca 2+ activation of crossbridge cycling (46).…”

“…We also studied the effect on Tm position of a shorter fragment (C0C1f; Fig. 1) known to bind to F-actin (16,27). Filaments decorated with C0C1f were wider than controls, clearly demonstrating binding of the fragment (Fig.…”

“…6, control). Although the shorter C0C2 fragment was used in our structural studies (to minimize interference with Tm visibility in the reconstructions), we have shown previously that C0C2 and C0C3 bind similarly to actin filaments (27) and are functionally identical in their inhibition of actin filament motility (16). On addition of 1 μM C0C3 (Fig.…”

“…18). In the in vitro motility assay, actin filament sliding over myosin is slowed by N-terminal fragments of cMyBP-C to the same extent as whole cMyBP-C (19), possibly by slowing the myosin detachment rate from actin (19) or tethering the thick to the thin filament (16,19). In an assay closer to the in vivo situation, the sliding of F-actin over native cardiac thick filaments was slowed specifically in the C-zone, and this slowing was ablated by removal of C0C1 and the first 17 amino acids of the M-domain [known as C0C1f (16); Fig.…”