Effect of extraction of myosin binding protein C on contractility of rat heart

Human hearts with reduced or mutant myosin binding protein C (MyBP-C) undergo hypertrophy and dilation, suggesting that reduction or alteration of MyBP-C interferes with normal contraction. Extraction of 60-70% of MyBP-C over 1 h from a mechanically disrupted cardiac myocyte has been shown to increase Ca sensitivity but does not appear to impair development of maximum Ca-activated force (Fmax). To determine whether loss of MyBP-C over a longer period of time will decrease force development in a reversible manner, MyBP-C has been extracted from chemically skinned rat cardiac trabeculae for 1-4 h, and force production, Ca sensitivity, and thick filament structure were measured. Although extraction of MyBP-C for 1 h did not alter Fmax, after 4 h, myosin heads became disordered and Fmax decreased. At this point, incubation of the trabeculae with rat cardiac MyBP-C in a relaxing solution reversed the decline in Fmax and most of the change in order of myosin heads. Extraction of MyBP-C appears to produce a change in the orientation of myosin heads that is associated with a decreased ability of the contractile system to develop force.     

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.     

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.     

A substitution mutation in the myosin binding protein C gene in ragdoll hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM) is a primary myocardial disease with a prevalence of 1 in 500 in human beings. Causative mutations have been identified in several sarcomeric genes, including the cardiac myosin binding protein C (MYBPC3) gene. Heritable HCM also exists in a large-animal model, the cat, and we have previously reported a mutation in the MYBPC3 gene in the Maine coon breed. We now report a separate mutation in the MYBPC3 gene in ragdoll cats with HCM. The mutation changes a conserved arginine to tryptophan and appears to alter the protein structure. The ragdoll is not related to the Maine coon and the mutation identified is in a domain different from that of the previously identified feline mutation. The identification of two separate mutations within this gene in unrelated breeds suggests that these mutations occurred independently rather than being passed on from a common founder.     

Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

Background  

Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA). Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs). Cardiac Myosin Binding Protein-C (cMyBPC) and cardiac troponin I (cTNI) are hypertrophic cardiomyopathy (HCM)-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation.     

Mapping the binding domain of a myosin II binding protein

The way in which actin and myosin II become localized to the contractile ring of dividing cells resulting in cleavage furrow formation and cytokinesis is unknown. While much is known about actin binding proteins and actin localization, little is known about myosin localization. A 53-kDa (53K) polypeptide present in the sea urchin egg binds to myosin II in a nucleotide-dependent manner and mediates its solubility in vitro [Yabkowitz, R., & Burgess, D.R. (1987) J. Cell Biol. 105, 927-936]. The binding site of 53K on the myosin molecule was examined in an effort to understand the mechanism of 53K-induced myosin solubility and its potential function in myosin regulation. Blot overlay and chemical cross-linking techniques utilizing myosin proteolytic fragments indicate that 53K binds to fragments proximal to the head-rod junction of myosin. Fragments distal to the head-rod junction do not bind 53K. In addition, the binding of 53K to myosin largely inhibits protease digestion that produces the head and rod fragments. The binding of 53K to the head-rod domain of myosin may be critical in regulation of myosin conformation, localization, assembly, and ATPase activity.     

Small-angle X-ray scattering reveals the N-terminal domain organization of cardiac myosin binding protein C

Myosin binding protein C (MyBP-C) is a multidomain accessory protein of striated muscle sarcomeres. Three domains at the N-terminus of MyBP-C (C1-m-C2) play a crucial role in maintaining and modulating actomyosin interactions. The cardiac isoform has an additional N-terminal domain (C0) that is postulated to provide a greater level of regulatory control in cardiac muscle. We have used small-angle X-ray scattering, ab initio shape restoration, and rigid-body modeling to determine the average shape and spatial arrangement of the four N-terminal domains of cardiac MyBP-C (C0C2) and a three-domain variant that is analogous to the N-terminus of the skeletal isoform (C1C2). We found that the domains of both proteins are tandemly arranged in a highly extended configuration that is sufficiently long to span the interfilament cross-bridge distances in vivo and, hence, be poised to modulate these interactions. The average spatial organization of the C1, m, and C2 domains is not significantly perturbed by the removal of the cardiac-specific C0 domain, suggesting that the interdomain interfaces, while relatively small in area, have a degree of rigidity. Modeling the C0C2 and C1C2 scattering data reveals that the structures of the C0 and m domains (also referred to as the ‘MyBP motif’) are compact and have dimensions that are consistent with the immunoglobulin fold superfamily of proteins. Sequence analysis, homology modeling, and circular dichroism experiments support the conclusion that the previously undetermined structures of these domains can be characterized as having an immunoglobulin-like fold. Atomic models using the known NMR structures for C1 and C2 as well as homology models for the C0 and m domains provide insights into the placement of conserved serine residues of the m domain that are phosphorylated in vivo and cause a change in muscle fiber contraction by abolishing interactions with myosin.     

The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C

Heat shock proteins play central roles in ensuring the correct folding and maturation of cellular proteins. Here we show that the heat shock protein Hsp70 has a novel role in prolonging the lifetime of activated protein kinase C. We identified Hsp70 in a screen for binding partners for the carboxyl terminus of protein kinase C. Co-immunoprecipitation experiments revealed that Hsp70 specifically binds the unphosphorylated turn motif (Thr(641) in protein kinase C beta II), one of three priming sites phosphorylated during the maturation of protein kinase C family members. The interaction of Hsp70 with protein kinase C can be abolished in vivo by co-expression of fusion proteins encoding the carboxyl terminus of protein kinase C or the carboxyl terminus of Hsp70. Pulse-chase experiments reveal that Hsp70 does not regulate the maturation of protein kinase C: the rate of processing by phosphorylation is the same in the presence or absence of disrupting constructs. Rather, Hsp70 prolongs the lifetime of mature protein kinase C; disruption of the interaction promotes the accumulation of matured and then dephosphorylated protein kinase C in the detergent-insoluble fraction of cells. Furthermore, studies with K562 cells reveal that disruption of the interaction with Hsp70 slows the protein kinase C beta II-mediated recovery of cells from PMA-induced growth arrest. Last, we show that other members of the AGC superfamily (Akt/protein kinase B and protein kinase A) also bind Hsp70 via their unphosphorylated turn motifs. Our data are consistent with a model in which Hsp70 binds the dephosphorylated carboxyl terminus of mature protein kinase C, thus stabilizing the protein and allowing re-phosphorylation of the enzyme. Disruption of this interaction prevents re-phosphorylation and targets the enzyme for down-regulation.     

Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties.

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.     

Computational analysis of folding and mutation properties of C5 domain of myosin binding protein C

Thermal folding molecular dynamics simulations of the domain C5 of Myosin binding protein C were performed using a native-centric model to study the role of three mutations related to Familial Hypertrophic Cardiomyopathy. Mutation of Asn755 causes the largest shift of the folding temperature, and the residue is located in the CFGA' beta-sheet featuring the highest phi-values. The mutation thus appears to reduce the thermodynamic stability in agreement with experimental data. The mutations on Arg654 and Arg668, conversely, cause little change in the folding temperature and they reside in the low phi-value BDE beta-sheet, so that their pathological role cannot be related to impairment of the folding process but possibly to the binding with target molecules. As the typical signature of Domain C5 is the presence of a longer and destibilizing CD-loop with respect to the other Ig-like domains, we completed the work with a bioinformatic analysis of this loop showing a high density of negative charge and low hydrophobicity. This indicates the CD-loop as a natively unfolded sequence with a likely coupling between folding and ligand binding.     

Analyzing pathogenic mutations of C5 domain from cardiac myosin binding protein C through MD simulations

The folding properties of wild type and mutants of domain C5 from cardiac myosin binding protein C have been investigated via molecular dynamics simulations within the framework of a native-centric and coarse-grained model. The relevance of a mutation has been assessed through the shift in the unfolding temperature, the change in the unfolding rate it determines and Phi-values analysis. In a previous paper (Guardiani et al. Biophys J 94:1403-1411, 2008), we performed Kinetic simulations on native contact formation revealing an entropy-driven folding pathway originating near the FG and DE loops. This folding mechanism allowed also a possible interpretation of the molecular impact of the three mutations, Arg14His, Arg28His and Asn115Lys involved in the Familial Hypertrophic Cardiomyopathy. Here we extend that analysis by enriching the mutant pool and we identify a correlation between unfolding rates and the number of native contacts retained in the transition state.     

Contribution of individual histidines to prion protein copper binding

The prion protein is well-established as a copper binding protein. The N-terminus of the protein contains an octameric repeat region with each of the four repeats containing a histidine. The N-terminus has two additional histidines distal to the repeat region that has been commonly known as the fifth site. While binding of copper by the protein has been extensively studied, the contribution of each histidine to copper binding in the full-length protein has not. Here we used a battery of mutants of the recombinant mouse prion protein to assess copper binding with both isothermal titration calorimetry and cyclic voltammetry. The findings indicate that there is extensive cooperativity between different binding sites in the protein. The two highest-affinity binding events occur at the fifth site and at the octameric repeat region. However, the first binding is that to the octameric repeat region. Subsequent binding events after the two initial binding events have lower affinities within the octameric repeat region.     

Stability and kinetic properties of C5-domain from myosin binding protein C and its mutants

The impact of three mutations of domain C5 from myosin binding protein C, correlated to Familial Hypertrophic Cardiomyopathy, has been assessed through molecular dynamics simulations based on a native centric protein modeling. The severity of the phenotype correlates with the shift in unfolding temperature determined by the mutations. A contact probability analysis reveals a folding process of the C5 domain originating in the region of DE and FG loops and propagating toward the area proximal to CD and EF loops. This suggests that mutation effects gain relevance in the proximity to the area where folding originates. The scenario is also confirmed by the analysis of the kinetics of 27 test mutations evenly distributed throughout the entire C5 domain.     

Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.

We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur.     

Domain formation in a fluid mixed lipid bilayer modulated through binding of the C2 protein motif

The role and mechanism of formation of lipid domains in a functional membrane have generally received limited attention. Our approach, based on the hypothesis that thermodynamic coupling between lipid-lipid and protein-lipid interactions can lead to domain formation, uses a combination of an experimental lipid bilayer model system and Monte Carlo computer simulations of a simple model of that system. The experimental system is a fluid bilayer composed of a binary mixture of phosphatidylcholine (PC) and phosphatidylserine (PS), containing 4% of a pyrene-labeled anionic phospholipid. Addition of the C2 protein motif (a structural domain found in proteins implicated in eukaryotic signal transduction and cellular trafficking processes) to the bilayer first increases and then decreases the excimer/monomer ratio of the pyrene fluorescence. We interpret this to mean that protein binding induces anionic lipid domain formation until the anionic lipid becomes saturated with protein. Monte Carlo simulations were performed on a lattice representing the lipid bilayer to which proteins were added. The important parameters are an unlike lipid-lipid interaction term and an experimentally derived preferential protein-lipid interaction term. The simulations support the experimental conclusion and indicate the existence of a maximum in PS domain size as a function of protein concentration. Thus, lipid-protein coupling is a possible mechanism for both lipid and protein clustering on a fluid bilayer. Such domains could be precursors of larger lipid-protein clusters ('rafts'), which could be important in various biological processes such as signal transduction at the level of the cell membrane.     

Contribution of myosin rod protein to the structural organization of adult and embryonic muscles in Drosophila

Myosin rod protein (MRP) is a naturally occurring 155 kDa protein in Drosophila that includes the myosin heavy chain (MHC) rod domain, but contains a unique 77 amino acid residue N-terminal region that replaces the motor and light chain-binding domains of S1. MRP is a major component of myofilaments in certain direct flight muscles (DFMs) and it is present in other somatic, cardiac and visceral muscles in adults, larvae and embryos, where it is coexpressed and polymerized into thick filaments along with MHC. DFM49 has a relatively high content of MRP, and is characterized by an unusually disordered myofibrillar ultrastructure, which has been attributed to lack of cross-bridges in the filament regions containing MRP. Here, we characterize in detail the structural organization of myofibrils in adult and embryonic Drosophila muscles containing various MRP/MHC ratios and in embryos carrying a null mutation for the single MHC gene. We examined MRP in embryonic body wall and intestinal muscles as well as in DFMs with consistent findings. In DFMs numbers 49, 53 and 55, MRP is expressed at a high level relative to MHC and is associated with disorder in the positioning of thin filaments relative to thick filaments in the areas of overlap. Embryos that express MRP in the absence of MHC form thick filaments that participate in the assembly of sarcomeres, suggesting that myofibrillogenesis does not depend on strong myosin-actin interactions. Further, although thick filaments are not well ordered, the relative positioning of thin filaments is fairly regular in MRP-only containing sarcomeres, confirming the hypothesis that the observed disorder in MRP/MHC containing wild-type muscles is due to the combined action between the functional behavior of MRP and MHC myosin heads. Our findings support the conclusion that MRP has an active function to modulate the contractile activity of muscles in which it is expressed.     

Identification of myosin II as a binding protein to the PH domain of protein kinase B

Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a Kd value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB.