The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner

Abstract

Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (K d ) of ϳ10 M and a molar binding ratio (B max ) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced B max ) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase crossbridge cycling. Cardiac myosin-binding protein C (cMyBP-C)2 is a thick filament accessory protein that performs both structural and regulatory functions within vertebrate sarcomeres. Both roles are likely to be essential in deciphering how a growing number of mutations found in the cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure in a substantial number of the world's population (1, 2).Considerable progress has recently been made in determining the regulatory functions of cMyBP-C and it is now apparent that cMyBP-C normally limits cross-bridge cycling kinetics and is critical for cardiac function (3-5). Phosphorylation of cMyBP-C is essential for its regulatory effects because elimination of phosphorylation sites (serine to alanine substitutions) abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge cycling kinetics and blunts cardiac responses to inotropic stimuli (6). The substitutions further impair cardiac function, reduce contractile reserve, and cause cardiac hypertrophy in transgenic mice (6, 7). By contrast, substitution of aspartic acids at these sites to mimic constitutive phosphorylation is benign or cardioprotective (8).Although a role for cMyBP-C in modulating cross-bridge kinetics is supported by several transgenic and knock-out mouse models (6, 7, 9, 10), the precise mechanisms by which cMyBP-C exerts these effects are not completely understood. For instance, the unique regulatory motif or "m-domain" of cMyBP-C binds to the S2 subfragment of myosin in vitro (11) and binding is abolished by PKA-mediated phosphorylation of the m-domain (12). These observations have led to the idea that (un)binding of the m-domain from myosin S2 mediates PKA-induced increases in cross-bridge cycling kinetics. Consistent with this idea, Calaghan and colleagues (13) showed that S2 added to transiently permeabilized myocytes increa...

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

“…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 note, finally, that cMyBP-C phosphorylation in response to beta-adrenergic stimulation leads to enhanced cardiac contractility (53). Serine phosphorylation in cMyBP-C's M-domain by a host of kinases reduces the affinity of the N terminus for actin (15) and myosin (7). Therefore, modulation of cMyBP-C's binding capacity by phosphorylation may add a measure of tunability to cMyBP-C's regulation of cardiac contractility in response to physiological stress.…”

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

“…An increasing number of results indicate that the N-terminal domains of cMyBP-C influence force activation through a direct interaction with actin. 71,[73][74][75] Data from small-angle solution scattering have provided new insights into the structure of cMyBP-C and its interactions with actin and how they might modulate the primary Ca 21 -signal.…”

Phosphorylation and calcium antagonistically tune myosin-binding protein C’s structure and function

“…β-Adrenergic-stimulated phosphorylation of these serines is believed to enhance cardiac contractility (11,12), and a high level of phosphorylation appears to be critical to normal cardiac function, whereas dephosphorylation has been associated with heart failure (13)(14)(15). Although the mechanistic role of cMyBP-C phosphorylation in vivo remains unclear, it is known to reduce the extensibility of the M-domain (10,16), diminish the binding of cMyBP-C to actin (17,18) and to myosin S2 (19), and to tune cMyBP-C's ability to modulate actomyosin activity in vitro (20)(21)(22)(23)(24).…”