C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

Abstract

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca 2+ have been achieved. We suggest that Ca 2+ modulates the interaction of cMyBP-C with the TF in the sarcomere.

Skeletal myosin binding protein-C: An increasingly important regulator of striated muscle physiology

“…However, the interaction of actin and MyBP-C remained largely unexplored until a series of landmark studies by the Harris group [34- 36]. The C0C2 domains bind the thin filament in a phosphorylation-dependent manner, and incubation with these domains results in a large increase in force production at submaximal Ca 2+ concentrations [35, 37, 38]. While the C0 domain can bind actin, it does not contribute to this phenomenon.…”

“…Instead, it seems that the C1 domain can bind to actin in a position that allows it to activate the thin filament by shifting tropomyosin. However, a direct interaction between tropomyosin and the proline-alanine-rich region of cMyBP-C may also contribute to this activation [37, 39]. …”

Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

“…Three of these sites are principally phosphorylated by protein kinase A (PKA) (10), whereas the fourth is principally phosphorylated by Ca 2+ -calmodulin-dependent kinase (CAMK) (10,32). When nonphosphorylated, binding of this region and associated N-terminal domains to both the S2-region of myosin (10,19,21,22,33,34) and F-actin (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) have been demonstrated. Phosphorylation of this region, in contrast, abolishes binding to myosin S2 and weakens the binding to F-actin (10,20,38,46,47).…”

“…Although much of the recent work on cMyBP-C has focused on its ability to bind to F-actin (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) as a possible regulatory mechanism, reversible phosphorylation-dependent binding of the N-terminal region of cMyBP-C to the S2-region of myosin (10, 20-23, 33, 34, 49) or to other regions of myosin (50,51) also remains a strong possibility as a mechanism by which cMyBP-C could regulate contraction.…”

Molecular Structure of Muscle Filaments Determined by Electron Microscopy

“…1E) should help to answer this question. Recent cryo-EM observations support different azimuthal positions of tropomyosin on actin in high resolution reconstructions (Harris et al, 2016; von der Ecken et al, 2015), suggesting that tropomyosin can indeed move; however, the low Ca 2+ position seen by negative staining is not well supported in these studies. At low Ca 2+ , tropomyosin appears to occupy the position seen at high Ca 2+ by negative stain, while at high Ca 2+ , tropomyosin is in the myosin-induced position (seen by negative stain).…”