Interaction of the C2 Ig-like Domain of Cardiac Myosin Binding Protein-C with F-actin

Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca²⁺ levels. Under activating conditions Ca²⁺ binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions. Recent studies have shown that in addition to Ca²⁺, the first four N-terminal domains (NTDs) of cardiac myosin binding protein C (cMyBP-C) (e.g. C0, C1, M and C2), are potent modulators of the TF activity, but the mechanism of their collective action is poorly understood. Previously, we showed that C1 activates the TF at low Ca²⁺ and C0 stabilizes binding of C1 to the TF, but the ability of C2 to bind and/or affect the TF remains unknown. Here we obtained 7.5 Å resolution cryo-EM reconstruction of C2-decorated actin filaments to demonstrate that C2 binds to actin in a single structural mode that does not activate the TF unlike the polymorphic binding of C0 and C1 to actin. Comparison of amino acid sequences of C2 with either C0 or C1 shows low levels of identity between the residues involved in interactions with the TF but high levels of conservation for residues involved in Ig fold stabilization. This provides a structural basis for strikingly different interactions of structurally homologous C0, C1 and C2 with the TF. Our detailed analysis of the interaction of C2 with the actin filament provides crucial information required to model the collective action of cMyBP-C NTDs on the cardiac TF.     

Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a “nanosurfer” assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human β-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0–C2 or C1–C2) on nanotubes every 14 nm. Both C0–C2 and C1–C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0–C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0–C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of β-cardiac myosin contractility.     

Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.     

Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C (cMyBP-C)

Myosin-binding protein C (MyBP-C) is an ∼ 130-kDa rod-shaped protein of the thick (myosin containing) filaments of vertebrate striated muscle. It is composed of 10 or 11 globular 10-kDa domains from the immunoglobulin and fibronectin type III families and an additional MyBP-C-specific motif. The cardiac isoform cMyBP-C plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon β-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart diseases. In addition to binding to myosin, MyBP-C can also bind to actin via its N-terminal end, potentially modulating contraction in a novel way via this thick-thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and three-dimensional reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25 to 180 mM KCl concentration. Three-dimensional helical reconstructions, carried out at the 180-mM KCl level to minimize nonspecific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin.     

Microscale thermophoresis suggests a new model of regulation of cardiac myosin function via interaction with cardiac myosin-binding protein C

The cardiac isoform of myosin-binding protein C (cMyBP-C) is a key regulatory protein found in cardiac myofilaments that can control the activation state of both the actin-containing thin and myosin-containing thick filaments. However, in contrast to thin filament–based mechanisms of regulation, the mechanism of myosin-based regulation by cMyBP-C has yet to be defined in detail. To clarify its function in this process, we used microscale thermophoresis to build an extensive interaction map between cMyBP-C and isolated fragments of β-cardiac myosin. We show here that the regulatory N-terminal domains (C0C2) of cMyBP-C interact with both the myosin head (myosin S1) and tail domains (myosin S2) with micromolar affinity via phosphorylation-independent and phosphorylation-dependent interactions of domain C1 and the cardiac-specific m-motif, respectively. Moreover, we show that the interaction sites with the highest affinity between cMyBP-C and myosin S1 are localized to its central domains, which bind myosin with submicromolar affinity. We identified two separate interaction regions in the central C2C4 and C5C7 segments that compete for the same binding site on myosin S1, suggesting that cMyBP-C can crosslink the two myosin heads of a single myosin molecule and thereby stabilize it in the folded OFF state. Phosphorylation of the cardiac-specific m-motif by protein kinase A had no effect on the binding of either the N-terminal or the central segments to the myosin head domain, suggesting this might therefore represent a constitutively bound state of myosin associated with cMyBP-C. Based on our results, we propose a new model of regulation of cardiac myosin function by cMyBP-C.     

Crystal structure of the C1 domain of cardiac myosin binding protein-C: implications for hypertrophic cardiomyopathy

C-protein is a major component of skeletal and cardiac muscle thick filaments. Mutations in the gene encoding cardiac C-protein [cardiac myosin binding protein-C (cMyBP-C)] are one of the principal causes of hypertrophic cardiomyopathy. cMyBP-C is a string of globular domains including eight immunoglobulin-like and three fibronectin-like domains termed C0-C10. It binds to myosin and titin, and probably to actin, and may have both a structural and a regulatory role in muscle function. To help to understand the pathology of the known mutations, we have solved the structure of the immunoglobulin-like C1 domain of MyBP-C by X-ray crystallography to a resolution of 1.55 Å. Mutations associated with hypertrophic cardiomyopathy are clustered at one end towards the C-terminus, close to the important C1C2 linker, where they alter the structural integrity of this region and its interactions.     

A Gain-of-Function Mutation in the M-domain of Cardiac Myosin-binding Protein-C Increases Binding to Actin

The M-domain is the major regulatory subunit of cardiac myosin-binding protein-C (cMyBP-C) that modulates actin and myosin interactions to influence muscle contraction. However, the precise mechanism(s) and the specific residues involved in mediating the functional effects of the M-domain are not fully understood. Positively charged residues adjacent to phosphorylation sites in the M-domain are thought to be critical for effects of cMyBP-C on cross-bridge interactions by mediating electrostatic binding with myosin S2 and/or actin. However, recent structural studies revealed that highly conserved sequences downstream of the phosphorylation sites form a compact tri-helix bundle. Here we used site-directed mutagenesis to probe the functional significance of charged residues adjacent to the phosphorylation sites and conserved residues within the tri-helix bundle. Results confirm that charged residues adjacent to phosphorylation sites and residues within the tri-helix bundle are important for mediating effects of the M-domain on contraction. In addition, four missense variants within the tri-helix bundle that are associated with human hypertrophic cardiomyopathy caused either loss-of-function or gain-of-function effects on force. Importantly, the effects of the gain-of-function variant, L348P, increased the affinity of the M-domain for actin. Together, results demonstrate that functional effects of the M-domain are not due solely to interactions with charged residues near phosphorylatable serines and provide the first demonstration that the tri-helix bundle contributes to the functional effects of the M-domain, most likely by binding to actin.     

Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.     

The C0C1 fragment of human cardiac myosin binding protein C has common binding determinants for both actin and myosin

The N-terminal domains of cardiac myosin binding protein C (MyBP-C) play a regulatory role in modulating interactions between myosin and actin during heart muscle contraction. Using NMR spectroscopy and small-angle neutron scattering, we have determined specific details of the interaction between the two-module human C0C1 cMyBP-C fragment and F-actin. The small-angle neutron scattering data show that C0C1 spontaneously polymerizes monomeric actin (G-actin) to form regular assemblies composed of filamentous actin (F-actin) cores decorated by C0C1, similar to what was reported in our earlier four-module mouse cMyBP-C actin study. In addition, NMR titration analyses show large intensity changes for a subset of C0C1 peaks upon addition of G-actin, indicating that human C0C1 interacts specifically with actin and promotes its assembly into filaments. During the NMR titration, peaks corresponding to cardiac-specific C0 domain are the first to be affected, followed by those from the C1 domain. No peak intensity or position changes were detected for peaks arising from the disordered proline/alanine-rich (P/A) linker connecting C0 with C1, despite previous suggestions of its involvement in binding actin. Of considerable interest is the observation that the actin-interaction “hot-spots” within the C0 and C1 domains, revealed in our NMR study, overlap with regions previously identified as binding to the regulatory light chain of myosin and to myosin ΔS2. Our results suggest that C0 and C1 interact with myosin and actin using a common set of binding determinants and therefore support a cMyBP-C switching mechanism between myosin and actin.     

The N-terminal domains of myosin binding protein C can bind polymorphically to F-actin

The regulation of vertebrate striated muscle contraction involves a number of different molecules, including the thin-filament accessory proteins tropomyosin and troponin that provide Ca²⁺-dependent regulation by controlling access to myosin binding sites on actin. Cardiac myosin binding protein C (cMyBP-C) appears to modulate this Ca²⁺-dependent regulation and has attracted increasing interest due to links with inherited cardiac diseases. A number of single amino acid mutations linked to clinical diseases occur in the N-terminal region of cMyBP-C, including domains C0 and C1, which previously have been shown to bind to F-actin. This N-terminal region also has been shown to both inhibit and activate actomyosin interactions in vitro. Using electron microscopy and three-dimensional reconstruction, we show that C0 and C1 can each bind to the same two distinctly different positions on F-actin. One position aligns well with the previously reported binding site that clashes with the binding of myosin to actin, but would force tropomyosin into an “on” position that exposes myosin binding sites along the filament. The second position identified here would not interfere with either myosin binding or tropomyosin positioning. It thus appears that the ability to bind to at least two distinctly different positions on F-actin, as observed for tropomyosin, may be more common than previously considered for other actin binding proteins. These observations help to explain many of the seemingly contradictory results obtained with cMyBP-C and show how cMyBP-C can provide an additional layer of regulation to actin-myosin interactions. They also suggest a redundancy of C0 and C1 that may explain the absence of C0 in skeletal muscle.     

Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C

Myosin binding protein-C (cMyBP-C) is a thick filament accessory protein, which in cardiac muscle functions to regulate the kinetics of cross-bridge interaction with actin; however, the underlying mechanism is not yet understood. To explore the structural basis for cMyBP-C function, we used synchrotron low-angle X-ray diffraction to measure interfilament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned myocardial preparations isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)). In relaxed myocardium, ablation of cMyBP-C appeared to result in radial displacement of cross-bridges away from the thick filaments, as there was a significant increase ( approximately 30%) in the I(11)/I(10) ratio for cMyBP-C(-/-) (0.37+/-0.03) myocardium as compared to WT (0.28+/-0.01). While lattice spacing tended to be greater in cMyBP-C(-/-) myocardium (44.18+/-0.68 nm) when compared to WT (42.95+/-0.43 nm), the difference was not statistically significant. Furthermore, liquid-like disorder in the myofilament lattice was significantly greater ( approximately 40% greater) in cMyBP-C(-/-) myocardium as compared to WT. These results are consistent with our working hypothesis that cMyBP-C normally acts to tether myosin cross-bridges nearer to the thick filament backbone, thereby reducing the likelihood of cross-bridge binding to actin and limiting cooperative activation of the thin filament.     

Human cardiac myosin binding protein C: structural flexibility within an extended modular architecture

New insights into the modular organization and flexibility of the N-terminal half of human cardiac myosin binding protein C (cMyBP-C) and information on the association state of the full-length protein have been deduced from a combined small-angle X-ray scattering (SAXS) and NMR study. SAXS data show that the first five immunoglobulin domains of cMyBP-C, which include those implicated in interactions with both myosin and actin, remain monodisperse and monomeric in solution and have a highly extended yet distinctively ‘bent’ modular arrangement that is similar to the giant elastic muscle protein titin. Analyses of the NMR and SAXS data indicate that a proline/alanine-rich linker connecting the cardiac-specific N-terminal C0 domain to the C1 domain provides significant structural flexibility at the N-terminus of the human isoform, while the modular arrangement of domains C1–C2–C3–C4 is relatively fixed. Domain fragments from the C-terminal half of the protein have a propensity to self-associate in vitro, while full-length bacterially expressed cMyBP-C forms flexible extended dimers at micromolar protein concentrations. In summary, our studies reveal that human cMyBP-C combines a distinctive modular architecture with regions of flexibility and that the N-terminal half of the protein is sufficiently extended to span the range of interfilament distances sampled within the dynamic environment of heart muscle. These structural features of cMyBP-C could facilitate its putative role as a molecular switch between actin and myosin and may contribute to modulating the transverse pliancy of the C-zone of the A-band across muscle sarcomeres.     

Binding of the N-terminal fragment C0-C2 of cardiac MyBP-C to cardiac F-actin

Cardiac myosin-binding protein C (cMyBP-C), a major accessory protein of cardiac thick filaments, is thought to play a key role in the regulation of myocardial contraction. Although current models for the function of the protein focus on its binding to myosin S2, other evidence suggests that it may also bind to F-actin. We have previously shown that the N-terminal fragment C0-C2 of cardiac myosin-binding protein-C (cMyBP-C) bundles actin, providing evidence for interaction of cMyBP-C and actin. In this paper we directly examined the interaction between C0-C2 and F-actin at physiological ionic strength and pH by negative staining and electron microscopy. We incubated C0-C2 (5-30μM, in a buffer containing in mM: 180 KCl, 1 MgCl(2), 1 EDTA, 1 DTT, 20 imidazole, at pH 7.4) with F-actin (5μM) for 30min and examined negatively-stained samples of the solution by electron microscopy (EM). Examination of EM images revealed that C0-C2 bound to F-actin to form long helically-ordered complexes. Fourier transforms indicated that C0-C2 binds with the helical periodicity of actin with strong 1st and 6th layer lines. The results provide direct evidence that the N-terminus of cMyBP-C can bind to F-actin in a periodic complex. This interaction of cMyBP-C with F-actin supports the possibility that binding of cMyBP-C to F-actin may play a role in the regulation of cardiac contraction.     

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

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 ∼10 μm 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 cross-bridge 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 increased their contractility, presumably because added S2 displaced cMyBP-C from binding endogenous S2. However, other reports indicate that cMyBP-C can influence actomyosin interactions through mechanisms unrelated to S2 binding, because either purified cMyBP-C (14) or recombinant N-terminal domains of cMyBP-C (15) affected acto-S1 filament sliding velocities and ATPase rates in the absence of myosin S2. These results thus raise the possibility that interactions with ligands other than myosin S2, such as actin or myosin S1, contribute to effects of cMyBP-C on cross-bridge interaction kinetics.The idea that cMyBP-C interacts with actin to influence cross-bridge cycling kinetics is supported by several studies that implicate the regulatory m-domain or sequences near it in actin binding (16-19). cMyBP-C is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 11 repeating domains that bear homology to either Ig or fibronectin-like folds. Domains are numbered sequentially from the N terminus of cMyBP-C as C0 through C10. The m-domain, a unique sequence of ∼100 amino acids, is located between domains C1 and C2 and is phosphorylated on at least 3 serine residues by PKA (12). Although the precise structure of the m-domain is not known, small angle x-ray scattering data suggest that it is compact and folded in solution and is thus similar in size and dimensions to the surrounding Ig domains (20). Recombinant proteins encompassing the m-domain and/or a combination of adjacent domains including C0, C1, C2, and a proline-alanine-rich sequence that links C0 to C1 have been shown to bind actin (16, 18, 19).The purpose of the present study was to characterize binding interactions of the N terminus of cMyBP-C with actin and to determine whether interactions with actin are influenced by phosphorylation of the m-domain. Results demonstrate that the N terminus of cMyBP-C binds to F-actin and to native thin filaments with affinities similar to that reported for cMyBP-C binding to myosin S2 (11). Furthermore, actin binding was reduced by m-domain phosphorylation, suggesting that reversible interactions of cMyBP-C with actin could contribute to modulation of cross-bridge kinetics.     

Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identification of a possible actin-binding domain

C-protein (MyBP-C) is a myosin-binding protein that is usually seen in two sets of seven to nine positions in the C-zones in each half of the vertebrate striated muscle A-band. Skeletal muscle C-protein is a modular structure containing ten sub-domains (C1 to C10) of which seven are immunoglobulin-type domains and three (C6, C7 and C9) are fibronectin-like domains. Cardiac muscle C-protein has an extra N-terminal domain (C0) and also some sequence insertions, one of which provides phosphorylation sites. It is conceivable that C-protein has both a structural and regulatory role within the sarcomere. The precise mode of binding of C-protein to the myosin filament has not been determined. However, detailed ultrastructural studies have suggested that C-protein, which binds to myosin, can give rise to a longer periodicity (about 435A) than the intrinsic myosin filament repeat of 429A. The reason for this has remained a puzzle for over 25 years. Here we show by modelling and computation that the presence of this longer periodicity could be explained if the myosin-binding part of C-protein binds to myosin with the expected 429A repeat, but if there are systematic interactions of the N-terminal end of C-protein with the neighbouring actin filaments in the hexagonal lattice of filaments in the A-band. We also show that if they occur these interactions would probably only arise in defined muscle states. Further analysis of the MyBP-C sequence identifies a possible actin-binding domain in the Pro-Ala-rich sequence found at the N terminus of skeletal MyBP-C and between domains C0 and C1 in the cardiac sequence.     

Multiple forms of cardiac myosin-binding protein C exist and can regulate thick filament stability

Although absence or abnormality of cardiac myosin binding protein C (cMyBP-C) produces serious structural and functional abnormalities of the heart, function of the protein itself is not clearly understood, and the cause of the abnormalities, unidentified. Here we report that a major function of cMyBP-C may be regulating the stability of the myosin-containing contractile filaments through phosphorylation of cMyBP-C. Antibodies were raised against three different regions of cMyBP-C to detect changes in structure within the molecule, and loss of myosin heavy chain was used to monitor degradation of the thick filament. Results from Western blotting and polyacrylamide gel electrophoresis indicate that cMyBP-C can exist in two different forms that produce, respectively, stable and unstable thick filaments. The stable form has well-ordered myosin heads and requires phosphorylation of the cMyBP-C. The unstable form has disordered myosin heads. In tissue with intact cardiac cells, the unstable unphosphorylated cMyBP-C is more easily proteolyzed, causing thick filaments first to release cMyBP-C and/or its proteolytic peptides and then myosin. Filaments deficient in cMyBP-C are fragmented by shear force well tolerated by the stable form. We hypothesize that modulation of filament stability can be coupled at the molecular level with the strength of contraction by the sensitivity of each to the concentration of calcium ions.     

The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility.     

E258K HCM-causing mutation in cardiac MyBP-C reduces contractile force and accelerates twitch kinetics by disrupting the cMyBP-C and myosin S2 interaction

Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C–S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.     

Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod

cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0-C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in beta myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a K(d) of 3.52 microM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.     

Effects of Cardiac Myosin Binding Protein-C on Actin Motility Are Explained with a Drag-Activation-Competition Model

Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin.