Human Cardiac Myosin Binding Protein Research Articles

Crystallisation and characterisation of muscle proteins: a mini-review.

The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of proteins. The most-commonly used method, however, remains X-ray crystallography but it does rely heavily on the production of suitable crystals. Indeed, the production of diffraction quality crystals remains the rate-limiting step for most protein systems. This mini-review highlights the crystallisation trials that used existing and newly developed crystallisation methods on two muscle protein targets - the actin binding domain (ABD) of α-actinin and the C0-C1 domain of human cardiac myosin binding protein C (cMyBP-C). Furthermore, using heterogenous nucleating agents the crystallisation of the C1 domain of cMyBP-C was successfully achieved in house along with preliminary actin binding studies using electron microscopy and co-sedimentation assays .

Human cardiac myosin-binding protein C phosphorylation- and mutation-dependent structural dynamics monitored by time-resolved FRET

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein of the sarcomere and a potential therapeutic target for treating contractile dysfunction in heart failure. Mimicking the structural dynamics of phosphorylated cMyBP-C by small-molecule drug binding could lead to therapies that modulate cMyBP-C conformational states, and thereby function, to improve contractility. We have developed a human cMyBP-C biosensor capable of detecting intramolecular structural changes due to phosphorylation and mutation. Using site-directed mutagenesis and time-resolved fluorescence resonance energy transfer (TR-FRET), we substituted cysteines in cMyBP-C N-terminal domains C0 through C2 (C0-C2) for thiol-reactive fluorescent probe labeling to examine C0-C2 structure. We identified a cysteine pair that upon donor-acceptor labeling reports phosphorylation-sensitive structural changes between the C1 domain and the tri-helix bundle of the M-domain that links C1 to C2. Phosphorylation reduced FRET efficiency by ~18%, corresponding to a ~11% increase in the distance between probes and a ~30% increase in disorder between them. The magnitude and precision of phosphorylation-mediated TR-FRET changes, as quantified by the Z'-factor, demonstrate the assay's potential for structure-based high-throughput screening of compounds for cMyBP-C-targeted therapies to improve cardiac performance in heart failure. Additionally, by probing C1's spatial positioning relative to the tri-helix bundle, these findings provide new molecular insight into the structural dynamics of phosphoregulation as well as mutations in cMyBP-C. Biosensor sensitivity to disease-relevant mutations in C0-C2 was demonstrated by examination of the hypertrophic cardiomyopathy mutation R282W. The results presented here support a screening platform to identify small molecules that regulate N-terminal cMyBP-C conformational states.

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.

Human cardiac myosin–binding protein C restricts actin structural dynamics in a cooperative and phosphorylation-sensitive manner

Cardiac myosin–binding protein C (cMyBP-C) is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner. To determine the effects of cMyBP-C and its phosphorylation on the microsecond rotational dynamics of actin filaments, we attached a phosphorescent probe to F-actin at Cys-374 and performed transient phosphorescence anisotropy (TPA) experiments. Binding of cMyBP-C N-terminal domains (C0–C2) to labeled F-actin reduced rotational flexibility by 20–25°, indicated by increased final anisotropy of the TPA decay. The effects of C0–C2 on actin TPA were highly cooperative (n = ∼8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers (i.e. the length of tropomyosin). PKA-mediated phosphorylation of C0–C2 eliminated the cooperative effects on actin flexibility and modestly increased actin rotational rates. Effects of Ser to Asp phosphomimetic substitutions in the M-domain of C0–C2 on actin dynamics only partially recapitulated the phosphorylation effects. C0–C1 (lacking M-domain/C2) similarly exhibited reduced cooperativity, but not as reduced as by phosphorylated C0–C2. These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. In contrast, phosphomimetic substitution of the glycogen synthase kinase (GSK3β) site in the Pro/Ala-rich linker of C0–C2 did not significantly affect the TPA results. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. We propose that these N-terminal cMyBP-C–induced changes in actin dynamics help explain the functional effects of cMyBP-C phosphorylation on actin-myosin interactions.

Intact-Mass Analysis Facilitating the Identification of Large Human Heart Proteoforms

Proteoforms, the primary effectors of biological processes, are the different forms of proteins that arise from molecular processing events such as alternative splicing and post-translational modifications. Heart diseases exhibit changes in proteoform levels, motivating the development of a deeper understanding of the heart proteoform landscape. Our recently developed two-dimensional top-down proteomics platform coupling serial size exclusion chromatography (sSEC) to reversed-phase chromatography (RPC) expanded coverage of the human heart proteome and allowed observation of high-molecular weight proteoforms. However, most of these observed proteoforms were not identified due to the difficulty in obtaining quality tandem mass spectrometry (MS2) fragmentation data for large proteoforms from complex biological mixtures on a chromatographic time scale. Herein, we sought to identify human heart proteoforms in this data set using an enhanced version of Proteoform Suite, which identifies proteoforms by intact mass alone. Specifically, we added a new feature to Proteoform Suite to determine candidate identifications for isotopically unresolved proteoforms larger than 50 kDa, enabling subsequent MS2 identification of important high-molecular weight human heart proteoforms such as lamin A (72 kDa) and trifunctional enzyme subunit α (79 kDa). With this new workflow for large proteoform identification, endogenous human cardiac myosin binding protein C (140 kDa) was identified for the first time. This study demonstrates the integration of our sSEC-RPC-MS proteomics platform with intact-mass analysis through Proteoform Suite to create a catalog of human heart proteoforms and facilitate the identification of large proteoforms in complex systems.

Differences in Myofilament Interactions and Structural Dynamics between Mouse and Human Cardiac Myosin-Binding Protein C

Differences in Myofilament Interactions and Structural Dynamics between Mouse and Human Cardiac Myosin-Binding Protein C

Structural Dynamics of Human Cardiac Myosin-Binding Protein C Revealed by Time-Resolved FRET

Structural Dynamics of Human Cardiac Myosin-Binding Protein C Revealed by Time-Resolved FRET

Structural characterization of the C3 domain of cardiac myosin binding protein C and its hypertrophic cardiomyopathy-related R502W mutant.

Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarcomere of cardiac muscle, regulates muscle contraction and is essential for proper muscle function. Hypertrophic cardiomyopathy (HCM), a genetic disease affecting 1 in 500 people, is the major cause of death in young athletes. It is caused by genetic mutations within sarcomeric proteins. Forty-two percent of the HCM-related mutations are found in cMyBP-C. Here we present the nuclear magnetic resonance-derived structural ensembles of the wild-type cMyBP-C C3 domain and its HCM-related R502W mutant. The C3 domain adopts an immunoglobulin-like fold, and mutation of the exposed Arg502 to a tryptophan does not perturb its structure, dynamics, or stability. However, the R502W mutation does alter the predicted electrostatic properties of the C3 domain. We hypothesize that this mutation, and other HCM-linked mutations found within the same domain, may directly disrupt the interaction of cMyBP-C with other sarcomeric proteins.

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.

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.

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.

Dissecting the N-terminal Myosin Binding Site of Human Cardiac Myosin-binding Protein C: STRUCTURE AND MYOSIN BINDING OF DOMAIN C2

Myosin-binding protein C (MyBP-C) binds to myosin with two binding sites, one close to the N terminus and the other at the C terminus. Here we present the solution structure of one part of the N-terminal binding site, the third immunoglobulin domain of the cardiac isoform of human MyBP-C (cC2) together with a model of its interaction with myosin. Domain cC2 has the beta-sandwich structure expected from a member of the immunoglobulin fold. The C-terminal part of the structure of cC2 is very closely related to telokin, the myosin binding fragment of myosin light chain kinase. Domain cC2 also contains two cysteines on neighboring strands F and G, which would be able to form a disulfide bridge in a similar position as in telokin. Using NMR spectroscopy and isothermal titration calorimetry we demonstrate that cC2 alone binds to a fragment of myosin, S2Delta, with low affinity (kD = 1.1 mM) but exhibits a highly specific binding site. This consists of the C-terminal surface of the C'CFGA' beta-sheet, which includes Glu(301), a residue mutated to Gln in the disease familial hypertrophic cardiomyopathy. The binding site on S2 was identified by a combination of NMR binding experiments of cC2 with S2Delta containing the cardiomyopathy-linked mutation R870H and molecular modeling. This mutation lowers the binding affinity and changes the arrangement of side chains at the interface. Our model of the cC2-S2Delta complex gives a first glimpse of details of the MyBP-C-myosin interaction. Using this model we suggest that most key interactions are between polar amino acids, explaining why the mutations E301Q in cC2 and R870H in S2Delta could be involved in cardiomyopathy. We expect that this model will stimulate future research to further refine the details of this interaction and their importance for cardiomyopathy.

WITHDRAWN: Disecting the N-terminal myosin binding site of human cardiac myosin binding protein C : Structure and myosin binding of domain C2

WITHDRAWN: Disecting the N-terminal myosin binding site of human cardiac myosin binding protein C : Structure and myosin binding of domain C2

117 Use of drosophila to approach the in vivo function of human cardiac myosin binding protein C

117 Use of drosophila to approach the in vivo function of human cardiac myosin binding protein C

Human cardiac myosin-binding protein C frameshift mutations result in marked decrease of protein expression

Human cardiac myosin-binding protein C frameshift mutations result in marked decrease of protein expression

COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes

Mutations in human cardiac myosin-binding protein C (cMyBP-C) gene are associated with familial hypertrophic cardiomyopathy (FHC), and most of them are predicted to produce COOH-truncated proteins. To understand the molecular mechanism(s) by which such mutations cause FHC, we analyzed (i) the accumulation of human cMyBP-C mutants in fetal rat cardiomyocytes, and (ii) the protein sequence of the human wild-type (wt) cMyBP-C by hydrophobic cluster analysis with the aim of identifying new putative myosin-binding site(s). Accumulation and sarcomeric localization of the wt protein and of four FHC-mutant cMyBP-Cs (E542Q and three COOH-truncated proteins) were studied in cardiomyocytes by immunostaining and confocal microscopy after transfection with myc-tagged constructs. We found that (i) 10 % of the cells expressing COOH-truncated mutants exhibit an incorporation into the A-band of the sarcomere without any alteration of the myofibrillar architecture versus 76 % of those expressing the wt or E542Q mutant cMyBP-Cs ( p < 0.001); (ii) 90 % of the cells expressing the truncated mutants show a diffuse localization of these proteins in the cardiomyocytes, out of which 45 % exhibit a significant alteration of the sarcomeric structure ( p < 0.0001 versus wt); and (iii) the two shortest mutant cMyBP-Cs accumulate at very low levels in fetal rat cardiomyocytes as compared to the wt ( p < 0.008). Protein sequence analysis indicated that a 45-residue sequence in the NH 2-terminal C0 domain of cMyBP-C exhibits a consistent homology (sequence similarity score of 42 %) with a segment of the NH 2-terminal domain of myomesin, another myosin-binding protein. This result suggests that the C0 domain of human cMyBP-C contains a novel putative myosin-binding site that could account for the A-band incorporation of the truncated mutants. In addition, the faint accumulation and the diffuse localization of truncated mutants could probably be explained by a low affinity of the C0 domain for myosin. We conclude that COOH-truncated cMyBP-Cs may act as poison polypeptides that disrupt the myofibrillar architecture and result in the defects observed in FHC.

Organization and Sequence of Human Cardiac Myosin Binding Protein C Gene (MYBPC3) and Identification of Mutations Predicted to Produce Truncated Proteins in Familial Hypertrophic Cardiomyopathy

Cardiac myosin binding protein C (MyBP-C) is a sarcomeric protein belonging to the intracellular immunoglobulin superfamily. Its function is uncertain, but for a decade evidence has existed for both structural and regulatory roles. The gene encoding cardiac MyBP-C (MYBPC3) in humans is located on chromosome 11p11.2, and mutations have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). Detailed characterization of the MYBPC3 gene is essential for studies on gene regulation, analysis of the role of MyBP-C in cardiac contraction through the use of recombinant DNA technology, and mutational analyses of FHC. The organization of human MYBPC3 and screening for mutations in a panel of French families with FHC were established using polymerase chain reaction, single-strand conformation polymorphism, and sequencing. The MYBPC3 gene comprises &gt;21 000 base pairs and contains 35 exons. Two exons are unusually small in size, 3 bp each. We found six new mutations associated with FHC in seven unrelated French families. Four of these mutations are predicted to produce truncated cardiac MyBP-C polypeptides. The two others should each produce two aberrant proteins, one truncated and one mutated. The present study provides the first organization and sequence for an MyBP-C gene. The mutations reported here and previously in MYBPC3 result in aberrant transcripts that are predicted to encode significantly truncated cardiac MyBP-C polypeptides. This spectrum of mutations differs from the ones previously observed in other disease genes causing FHC. Our data strengthen the functional importance of MyBP-C in the regulation of cardiac work and provide the basis for further studies.