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.     

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.     

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.     

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.     

Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca²⁺ levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.     

Role of actin C-terminus in regulation of striated muscle thin filament

In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca²⁺ and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca²⁺, but it reduces tropomyosin-troponin affinity in the absence of Ca²⁺. In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca²⁺-dependent regulation of the ATPase activity is preserved. Without Ca²⁺ the ATPase activity is fully inhibited, but in the presence of Ca²⁺ the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation.     

A high-throughput fluorescence lifetime-based assay to detect binding of myosin-binding protein C to F-actin

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin''s Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

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.     

Three-Dimensional Organization of Troponin on Cardiac Muscle Thin Filaments in the Relaxed State

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca²⁺ sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.     

Involvement of an arginine residue of actin in tropomyosin binding.

Using 1,2-cyclohexanedione, modification of three arginines per actin monomer in F-actin resulted in a loss of ability of the actin to interact with tropomyosin, although the F-actin polymer was not significantly depolymerized, the ability of the actin to activate the Mg²⁺-ATPase of myosin was not affected, and the secondary structure of the actin monomers was not appreciably altered. Isolation of peptides from a digest of modified F-actin indicated that the modified residues were Arg-28, Arg-95 and Arg-147. When actin was combined with tropomyosin prior to the modification treatment, Arg-95 was not modified, and the actin retained its ability to bind tropomyosin. These results therefore indicate a direct involvement of Arg-95 in the tropomyosin binding function of F-actin.     

The regulatory effects of tropomyosin and troponin-I on the interaction of myosin loop regions with F-actin

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088-5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac beta-myosin, presumed to be sites of interaction with F-actin (residues 554-584, 622-646, and 633-660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134-147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.     

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.     

Three-Dimensional Organization of Troponin on Cardiac Muscle Thin Filaments in the Relaxed State

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca²⁺ sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.     

The Motif of Human Cardiac Myosin-binding Protein C Is Required for Its Ca2+-dependent Interaction with Calmodulin

The N-terminal modules of cardiac myosin-binding protein C (cMyBP-C) play a regulatory role in mediating interactions between myosin and actin during heart muscle contraction. The so-called "motif," located between the second and third immunoglobulin modules of the cardiac isoform, is believed to modulate contractility via an "on-off" phosphorylation-dependent tether to myosin ΔS2. Here we report a novel Ca(2+)-dependent interaction between the motif and calmodulin (CaM) based on the results of a combined fluorescence, NMR, and light and x-ray scattering study. We show that constructs of cMyBP-C containing the motif bind to Ca(2+)/CaM with a moderate affinity (K(D) ～10 μm), which is similar to the affinity previously determined for myosin ΔS2. However, unlike the interaction with myosin ΔS2, the Ca(2+)/CaM interaction is unaffected by substitution with a triphosphorylated motif mimic. Further, Ca(2+)/CaM interacts with the highly conserved residues (Glu(319)-Lys(341)) toward the C-terminal end of the motif. Consistent with the Ca(2+) dependence, the binding of CaM to the motif is mediated via the hydrophobic clefts within the N- and C-lobes that are known to become more exposed upon Ca(2+) binding. Overall, Ca(2+)/CaM engages with the motif in an extended clamp configuration as opposed to the collapsed binding mode often observed in other CaM-protein interactions. Our results suggest that CaM may act as a structural conduit that links cMyBP-C with Ca(2+) signaling pathways to help coordinate phosphorylation events and synchronize the multiple interactions between cMyBP-C, myosin, and actin during the heart muscle contraction.     

Troponin Revealed: Uncovering the Structure of the Thin Filament On-Off Switch in Striated Muscle

Recently, our understanding of the structural basis of troponin-tropomyosin’s Ca²⁺-triggered regulation of striated muscle contraction has advanced greatly, particularly via cryo-electron microscopy data. Compelling atomic models of troponin-tropomyosin-actin were published for both apo- and Ca²⁺-saturated states of the cardiac thin filament. Subsequent electron microscopy and computational analyses have supported and further elaborated the findings. Per cryo-electron microscopy, each troponin is highly extended and contacts both tropomyosin strands, which lie on opposite sides of the actin filament. In the apo-state characteristic of relaxed muscle, troponin and tropomyosin hinder strong myosin-actin binding in several different ways, apparently barricading the actin more substantially than does tropomyosin alone. The troponin core domain, the C-terminal third of TnI, and tropomyosin under the influence of a 64-residue helix of TnT located at the overlap of adjacent tropomyosins are all in positions that would hinder strong myosin binding to actin. In the Ca²⁺-saturated state, the TnI C-terminus dissociates from actin and binds in part to TnC; the core domain pivots significantly; the N-lobe of TnC binds specifically to actin and tropomyosin; and tropomyosin rotates partially away from myosin’s binding site on actin. At the overlap domain, Ca²⁺ causes much less tropomyosin movement, so a more inhibitory orientation persists. In the myosin-saturated state of the thin filament, there is a large additional shift in tropomyosin, with molecular interactions now identified between tropomyosin and both actin and myosin. A new era has arrived for investigation of the thin filament and for functional understandings that increasingly accommodate the recent structural results.     

Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin.

Polarized phosphorescence from the triplet probe erythrosin-5-iodoacetamide attached to sulfhydryls in rabbit skeletal and cardiac muscle tropomyosin (Tm) was used to measure the microsecond rotational dynamics of these tropomyosins in a complex with F-actin. The steady-state phosphorescence anisotropy of skeletal tropomyosin on F-actin was 0.025 +/- 0.005 at 20 degrees C; the comparable anisotropy for cardiac tropomyosin was 0.010 +/- 0. 003. Measurements of the anisotropy as a function of temperature and solution viscosity (modulated by addition of glycerol) indicated that both skeletal and cardiac tropomyosin undergo complex rotational motions on the surface of F-actin. Models assuming either long axis rotation of a rigid rod or torsional twisting of a flexible rod adequately fit these data; both analyses indicated that cardiac Tm is more mobile than skeletal Tm and that the increased mobility on the surface of F-actin reflected either the rotational motion of a smaller physical unit or the torsional twisting of a less rigid molecule. The binding of myosin heads (S1) to the Tm-F-actin complexes increased the anisotropy to 0.049 +/- 0.004 for skeletal and 0.054 +/- 0.007 for cardiac tropomyosin. The titration of the skeletal tropomyosin-F-actin complex by S1 showed a break at an S1/actin ratio of 0.14; this complex had an anisotropy of 0.040 +/- 0.007, suggesting that one bound head effectively restricted the motion of each skeletal tropomyosin. A similar titration with cardiac tropomyosin reached a plateau at an S1/actin ratio of 0.4, suggesting that 2-3 myosin heads are required to immobilize cardiac Tm. Surface mobility is predicted by structural models of the interaction of tropomyosin with the actin filament while the decrease in tropomyosin mobility upon S1 binding is consistent with current theories for the proposed role of myosin binding in the mechanism of tropomyosin-based regulation of muscle 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.     

Effects of the N-terminal domains of myosin binding protein-C in an in vitro motility assay: Evidence for long-lived cross-bridges

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.     

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.