RESUMEN
Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by a ventricular hypertrophy predominantly affecting the interventricular septum and associated with a large extent of myocardial and myofibrillar disarray. It is the most common cause of sudden death in the young. In the four disease loci found, three genes have been identified which code for beta-myosin heavy chain, cardiac troponin T and alpha-tropomyosin. Recently the human cardiac myosin binding protein-C (MyBP-C) gene was mapped to chromosome 11p11.2 (ref. 8), making this gene a good candidate for the fourth locus, CMH4 (ref. 5). Indeed, MyBP-C is a substantial component of the myofibrils that interacts with several proteins of the thick filament of the sarcomere. In two unrelated French families linked to CMH4, we found a mutation in a splice acceptor site of the MyBP-C gene, which causes the skipping of the associated exon and could produce truncated cardiac MyBP-Cs. Mutations in the cardiac MyBP-C gene likely cause chromosome 11-linked hypertrophic cardiomyopathy, further supporting the hypothesis that hypertrophic cardiomyopathy results from mutations in genes encoding contractile proteins.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Proteínas Portadoras/genética , Mutación/genética , Empalme del ARN , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Humanos Par 11 , Femenino , Ligamiento Genético , Haplotipos , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteínas Musculares/química , Proteínas Musculares/fisiología , Sarcómeros/química , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Western Blotting , Calmodulina/metabolismo , Adhesión Celular , Células Cultivadas , Embrión de Pollo , Cromosomas Humanos Par 1 , Clonación Molecular , ADN Complementario/metabolismo , Epítopos , Biblioteca de Genes , Humanos , Inmunoglobulinas/metabolismo , Microscopía Confocal , Modelos Genéticos , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Filogenia , Unión Proteica , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Factores de Intercambio de Guanina Nucleótido Rho , Homología de Secuencia de Aminoácido , Transducción de Señal , Distribución Tisular , TransfecciónRESUMEN
In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Proteínas Quinasas/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Animales , Anticuerpos/inmunología , Células Cultivadas , Pollos , Conectina , Elasticidad , Epítopos/inmunología , Microscopía Inmunoelectrónica , Modelos Biológicos , Proteínas Motoras Moleculares/metabolismo , Proteínas Musculares/química , Proteínas Musculares/genética , Miocardio/citología , Miocardio/ultraestructura , Miofibrillas/ultraestructura , Miosinas/metabolismo , Pliegue de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/genética , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Sarcómeros/metabolismo , Sarcómeros/ultraestructura , TransfecciónRESUMEN
The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated approximately 52 nm from the central M1-line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches approximately 60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central M1-line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M-protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the M1-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.
Asunto(s)
Proteínas Musculares/análisis , Proteínas de Mieloma , Proteínas Quinasas/análisis , Sarcómeros/química , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Proteínas de Unión a Calmodulina/análisis , Bovinos , Conectina , Citoesqueleto/química , Mapeo Epitopo , Microscopía Inmunoelectrónica , Estructura Molecular , Proteínas Musculares/inmunología , Proteínas Quinasas/inmunología , Estructura Terciaria de Proteína , Conejos , Ratas , Sarcómeros/ultraestructuraRESUMEN
Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.
Asunto(s)
Inmunoglobulinas/química , Proteínas Musculares/química , Pliegue de Proteína , Proteínas Quinasas/química , Adsorción , Conectina , Elasticidad , Entropía , Microscopía de Fuerza Atómica , Método de Montecarlo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Estrés Mecánico , TermodinámicaRESUMEN
Familial hypertrophic cardiomyopathy is a disease generally believed to be caused by mutations in sarcomeric proteins. In a family with hypertrophic cardiomyopathy linked to polymorphic markers on chromosome 11, we found a new mutation of a splice donor site of the cardiac myosin-binding protein-C gene. This mutation causes the skipping of the associated exon in mRNA from lymphocytes and myocardium. Skipping of the exon with a consecutive reading frame shift leads to premature termination of translation and is thus expected to produce a truncated cardiac myosin-binding protein-C with loss of the myosin- and titin-binding COOH terminus. However, Western blot analysis of endomyocardial biopsies from histologically affected left ventricular myocardium failed to show the expected truncated protein. These data show for the first time that a splice donor site mutation in the myosin-binding protein-C gene is transcribed to cardiac mRNA. Truncated cardiac myosin-binding protein-C does not act as a "poison polypeptide," since it seems not to be incorporated into the sarcomere in significant amounts. The absence of mutant protein and of significantly reduced amounts of wild-type protein in the presence of the mutated mRNA argues against the "poison protein" and the "null allele" hypotheses and suggests yet unknown mechanisms relevant to the genesis of chromosome-11- associated familial hypertrophic cardiomyopathy.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Proteínas Portadoras/genética , Mutación , Miocardio/química , Empalme del ARN/genética , Western Blotting , Cardiomiopatía Hipertrófica/metabolismo , Proteínas Portadoras/análisis , Cromosomas Humanos Par 11/genética , Electroforesis en Gel de Poliacrilamida , Femenino , Ligamiento Genético , Humanos , Masculino , Linaje , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Polimorfismo Conformacional Retorcido-Simple , Eliminación de SecuenciaRESUMEN
The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca(2+)] reversed the PEVK effect. PEVK concentrations >/=10 microgram/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Miofibrillas/metabolismo , Proteínas Quinasas/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Unión Competitiva/fisiología , Bioensayo , Pollos , Conectina , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Proteínas Musculares/genética , Contracción Miocárdica/fisiología , Unión Proteica/fisiología , Proteínas Quinasas/genética , Estructura Terciaria de Proteína/fisiología , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcómeros/fisiología , Estrés Mecánico , Temperatura , ViscosidadRESUMEN
BACKGROUND: The giant muscle protein titin contributes to the filament system in skeletal and cardiac muscle cells by connecting the Z disk and the central M line of the sarcomere. One of the physiological functions of titin is to act as a passive spring in the sarcomere, which is achieved by the elastic properties of its central I band region. Titin contains about 300 domains of which more than half are folded as immunoglobulin-like (Ig) domains. Ig domain segments of the I band of titin have been extensively used as templates to investigate the molecular basis of protein elasticity. RESULTS: The structure of the Ig domain I1 from the I band of titin has been determined to 2.1 A resolution. It reveals a novel, reversible disulphide bridge, which is neither required for correct folding nor changes the chemical stability of I1, but it is predicted to contribute mechanically to the elastic properties of titin in active sarcomeres. From the 92 Ig domains in the longest isoform of titin, at least 40 domains have a potential for disulphide bridge formation. CONCLUSIONS: We propose a model where the formation of disulphide bridges under oxidative stress conditions could regulate the elasticity of the I band in titin by increasing sarcomeric resistance. In this model, the formation of the disulphide bridge could refrain a possible directed motion of the two beta sheets or other mechanically stable entities of the I1 Ig domain with respect to each other when exposed to mechanical forces.
Asunto(s)
Disulfuros/química , Disulfuros/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculos/química , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Conectina , Cristalografía por Rayos X , Elasticidad , Humanos , Inmunoglobulinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Espectrometría de Fluorescencia , Electricidad EstáticaRESUMEN
BACKGROUND: Hypertrophic cardiomyopathy is a myocardial disorder resulting from inherited sarcomeric dysfunction. We report a mutation in the myosin-binding protein-C (MyBP-C) gene, its clinical consequences in a large family, and myocardial tissue findings that may provide insight into the mechanism of disease. METHODS AND RESULTS: History and clinical status (examination, ECG, and echocardiography) were assessed in 49 members of a multigeneration family. Linkage analysis implicated the MyBP-C gene on chromosome 11. Myocardial mRNA, genomic MyBP-C DNA, and the myocardial proteins of patients and healthy relatives were analyzed. A single guanine nucleotide insertion in exon 25 of the MyBP-C gene resulted in the loss of 40 bases in abnormally processed mRNA. A 30-kDa truncation at the C-terminus of the protein was predicted, but a polypeptide of the expected size ( approximately 95 kDa) was not detected by immunoblot testing. The disease phenotype in this family was characterized in detail: only 10 of 27 gene carriers fulfilled diagnostic criteria. Five carriers showed borderline hypertrophic cardiomyopathy, and 12 carriers were asymptomatic, with normal ECG and echocardiograms. The age of onset in symptomatic patients was late (29 to 68 years). In 2 patients, outflow obstruction required surgery. Two family members experienced premature sudden cardiac death, but survival at 50 years was 95%. CONCLUSIONS: Penetrance of this mutation was incomplete and age-dependent. The large number of asymptomatic carriers and the good prognosis support the interpretation of benign disease.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Proteínas Portadoras/genética , Penetrancia , Adulto , Edad de Inicio , Anciano , Cardiomiopatía Hipertrófica/diagnóstico , Cromosomas Humanos Par 11 , Ecocardiografía , Electrocardiografía , Exones , Femenino , Ligamiento Genético , Genotipo , Heterocigoto , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Mutación , FenotipoRESUMEN
The myosin filaments of striated muscle contain a family of enigmatic myosin-binding proteins (MyBP), MyBP-C and MyBP-H. These modular proteins of the intracellular immunoglobulin superfamily contain unique domains near their N termini. The N-terminal domain of cardiac MyBP-C, the MyBP-C motif, contains additional phosphorylation sites and may regulate contraction in a phosphorylation dependent way. In contrast to the C terminus, which binds to the light meromyosin portion of the myosin rod, the interactions of this domain are unknown. We demonstrate that fragments of MyBP-C containing the MyBP-C motif localise to the sarcomeric A-band in cardiomyocytes and isolated myofibrils, without affecting sarcomere structure. The binding site for the MyBP-C motif resides in the N-terminal 126 residues of the S2 segment of the myosin rod. In this region, several mutations in beta-myosin are associated with FHC; however, their molecular implications remained unclear. We show that two representative FHC mutations in beta-myosin S2, R870H and E924K, drastically reduce MyBP-C binding (Kd approximately 60 microM for R870H compared with a Kd of approximately 5 microM for the wild-type) down to undetectable levels (E924K). These mutations do not affect the coiled-coil structure of myosin. We suggest that the regulatory function of MyBP-C is mediated by the interaction with S2, and that mutations in beta-myosin S2 may act by altering the interactions with MyBP-C.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Proteínas Portadoras/metabolismo , Mutación/genética , Miosinas/genética , Sitios de Unión/genética , Calorimetría , Técnicas de Cultivo de Célula , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Miosinas/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Conformación Proteica , Estructura Secundaria de Proteína , Sarcómeros/metabolismo , Alineación de Secuencia , UltracentrifugaciónRESUMEN
Titin has been suggested to act as a molecular ruler for the precise assembly of thick filaments in vertebrate striated muscle. To investigate the correlation of titin domain patterns with the architecture of the thick filament at its end, we have investigated the axial position of titin epitopes at the thick-filament/I-band junction. Antibodies against immunoglobin (Ig) domains N and C-terminal to the unique block of six fibronectin-3 (fn3) domains in this region were used. The distance between these epitopes confirms the idea that titin is laid out linearly along the thick filament with each domain measuring about 4 nm in length. Our data demonstrate that the gap of myosin crossbridges near the end of the thick filament closely correlates with the stretch of six fn3 domains, and that the last two crossbridges are at the level of the first two groups of fn3 domains which were previously assigned to the I-band. We conclude that the pattern of groups of fn3 domains reflects the arrangement of the myosin heads, at least at the end of the A-band. It seems likely that an alteration in the interaction between myosin and the titin fn3 domains towards the end of the thick filament is important for the formation of the crossbridge gap and thus the termination of the thick filament.
Asunto(s)
Citoesqueleto de Actina/fisiología , Proteínas Musculares/fisiología , Músculo Esquelético/fisiología , Miosinas/fisiología , Proteínas Quinasas/fisiología , Animales , Sitios de Unión , Conectina , Mapeo Epitopo , Fibronectinas/inmunología , Humanos , Datos de Secuencia Molecular , Músculo Esquelético/ultraestructura , Miofibrillas/fisiología , Miofibrillas/ultraestructura , Papio , Músculos Psoas , Conejos , Sarcómeros/fisiología , Sarcómeros/ultraestructuraRESUMEN
The huge modular protein nebulin is located in the thin filament of striated muscle in vertebrates and is thought to bind and stabilize F-actin. The C-terminal part of human nebulin is anchored in the sarcomeric Z-disk and contains an SH3 domain, the first of such motifs to be identified in a myofibrillar protein. We have determined the nebulin SH3 sequence from several species and found it strikingly conserved. We have also shown that the SH3 transcripts are constitutively expressed in skeletal muscle tissues. As the first step towards a molecular understanding of nebulin's cellular role we have determined the three-dimensional structure of the human nebulin SH3 domain in solution by nuclear magnetic resonance (NMR) spectroscopy and compared it with other known SH3 structures. The nebulin SH3 domain has a well-defined structure in solution with a typical SH3 topology, consisting of a beta-sandwich of two triple-stranded, antiparallel beta-sheets arranged at right angles to each other and of a single turn of a 310-helix. An additional double-stranded antiparallel beta-sheet in the RT loop bends over the beta-sandwich. The derived structure reveals a remarkable similarity with a distinct subset of SH3 domains, especially in the structural features of the exposed hydrophobic patch that is thought to be the site of interaction with polyproline ligands. On the basis of this similarity, we have modelled the interaction with an appropriate polyproline ligand and attempted to delineate the characteristics of the physiological SH3-binding partner in the Z-disk. Our results represent the first step in reconstructing the structure of nebulin and are expected to contribute to our understanding of nebulin's functional role in myofibrillar assembly.
Asunto(s)
Proteínas Musculares/química , Conformación Proteica , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conejos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Soluciones , Relación Estructura-ActividadRESUMEN
The vertebrate striated muscle protein titin is thought to play a critical rôle in myofibril assembly and passive tension. The recently determined complete primary structure of titin revealed a modular architecture that opens the way to a structural characterisation and the understanding of essential properties of this molecule through dissection into units that are structurally and/or functionally relevant. To understand the assembly process of titin, and ultimately the molecular basis of its elastic behaviour, we studied the thermodynamic properties of module pairs, the smallest structural unit that includes a module-module interface. Thus, selected module pairs and their component single modules from the I-band part of the titin molecule were expressed in Escherichia coli and their heat-induced and denaturant-induced unfolding was investigated with a combination of techniques (circular dichroism, fluorescence spectroscopy and nuclear magnetic resonance). The stabilities of single modules and pairs were determined from denaturation experiments. The module interface was also modelled on the basis of the sequence alignment of all approximately 40 immunoglobulin like modules from the I-band and the known structure of one of them. Our results show that all modules and module pairs examined are independently folded in solution. When covalently linked, although weakly interacting, they still behave as autonomous co-operative units upon unfolding. These observations lead us to suggest that folding of titin in vitro is a hierarchical event and that weak interactions between its adjacent modules must only partly account for its presumed elastic function.
Asunto(s)
Proteínas Musculares/química , Proteínas Quinasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dicroismo Circular , Conectina , Cartilla de ADN , Calefacción , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proteínas Musculares/efectos de los fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Conejos , Espectrometría de Fluorescencia , Urea/farmacologíaRESUMEN
Calmodulin (CaM)-protein interactions are usually described by studying complexes between synthetic targets of ca 25 amino acids and CaM. To understand the relevance of contacts outside the protein-binding region, we investigated the complex between recombinant human CaM (hCaM) and P7, a 38-residue peptide corresponding to the autoinhibitory domain of human cardiac titin kinase (hTK). To expedite the structure determination of hCaM-P7 we relied upon the high degree of similarity with other CaM-kinase peptide complexes. By using a combined homonuclear NMR spectroscopy and molecular modeling approach, we verified for the bound hCaM similar trends in chemical shifts as well as conservation of NOE patterns, which taken together imply the conservation of CaM secondary structure. P7 was anchored to the protein with 52 experimental intermolecular contacts. The hCaM-P7 structure is very similar to known CaM complexes, but the presence of NOE contacts outside the binding cavity appears to be novel. Comparison with the hTK crystal structure indicates that the P7 charged residues all correspond to accessible side-chains, while the putative anchoring hydrophobic side-chains are partially buried. To test this finding, we also modeled the early steps of the complex formation between Ca(2+)-loaded hCaM and hTK. The calculated trajectories strongly suggest the existence of an "electrostatic funnel", driving the long-range recognition of the two proteins. On the other hand, on a nanosecond time scale, no intermolecular interaction is formed as the P7 hydrophobic residues remain buried inside hTK. These results suggest that charged residues in hTK might be the anchoring points of Ca(2+)/hCaM, favoring the intrasteric regulation of the kinase. Furthermore, our structure, the first of CaM bound to a peptide derived from a kinase whose three-dimensional structure is known, suggests that special care is needed in the choice of template peptides to model protein-protein interactions.
Asunto(s)
Calmodulina/química , Calmodulina/metabolismo , Modelos Moleculares , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Conectina , Humanos , Datos de Secuencia Molecular , Proteínas Musculares/química , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Quinasas/química , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.
Asunto(s)
Elasticidad , Inmunoglobulinas/análisis , Proteínas Musculares/química , Músculos/fisiología , Proteínas Quinasas/química , Conectina , Espectroscopía de Resonancia Magnética , Conformación Proteica , Proteínas Recombinantes/química , Dispersión de RadiaciónRESUMEN
Titin is amongst the first sarcomeric proteins to be detected in the process of myofibrillogenesis of striated muscle. During embryogenesis this high molecular weight protein is initially observed in a punctate staining pattern in immunohistochemical studies, while during maturation titin organizes into a cross-striated pattern. The dynamic process of titin assembly up to its integration into the sarcomeres of cultured human skeletal muscle cells has been studied in subsequent stages of differentiation with antibodies to four well-defined titin epitopes. Since in maturated muscle cells these epitopes are clearly distinguishable on the extended titin molecule we wondered how these epitopes reorganize during myofibrillogenesis, and whether such a reorganization would reveal important clues about its supramolecular organization during development. Immunofluorescence staining of postmitotic mononuclear myoblasts indicate that the investigated epitopes of the titin molecule are displayed in a punctate pattern with neighboring, but clearly separate spots in the cytoplasm of the cells. During elongation and fusion of the cells, these titin spots associate with stress fiber-like structures to finally reach their position at either the Z-line, the A-I junction or the A-band. We propose that during this transition the large titin molecule is unfolded, with the amino terminus of the molecule migrating in the direction of the Z-line and the carboxy terminus moving towards the M-line. In maturated, fused myotubes the final cross-striated patterns of all investigated titin epitopes are observed. While this process of unfolding of the titin molecule progresses, other compounds of the Z-line and the A-band migrate to their specific positions in the nascent sarcomere. A-band components such as sarcomeric myosin and C-protein, are also observed as dot-like aggregates during initial stages of muscle cell differentiation and organize into a cross-striated pattern in the sarcomere virtually simultaneously with titin. The Z-line associated component desmin organizes into a cross-striated pattern at a later stage.
Asunto(s)
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinasas/metabolismo , Sarcómeros/metabolismo , Adulto , Animales , Diferenciación Celular , Células Cultivadas , Conectina , Humanos , Ratones , Mitosis , Músculo Esquelético/citologíaRESUMEN
In the I-band of skeletal muscle sarcomeres, the elastic region of titin consists of immunoglobulin (Ig) domains, and non-modular regions rich in proline, hydrophobic, and charged residues (PEVK). Using immunoelectron microscopy with sequence-assigned monoclonal antibodies, we demonstrate that extension of the Ig regions in M. psoas occurs largely at sarcomere lengths between 2 and 2.8 micron, decreasing in slope towards higher lengths. The Ig domains do not unfold. Above 2.6 micron, length changes are increasingly due to the PEVK-rich regions. We therefore propose that rubber-like properties of the PEVK-rich regions are mainly contributing to skeletal titin elasticity.
Asunto(s)
Proteínas Musculares/química , Proteínas Quinasas/química , Estructura Terciaria de Proteína , Sarcómeros/química , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Anticuerpos Monoclonales , Conectina , Elasticidad , Mapeo Epitopo/métodos , Inmunoglobulinas/química , Datos de Secuencia Molecular , Pliegue de Proteína , Músculos Psoas , Conejos , Sarcómeros/ultraestructuraRESUMEN
Myosin binding protein C is a protein of the myosin filaments of striated muscle which is expressed in isoforms specific for cardiac and skeletal muscle. The cardiac isoform is phosphorylated rapidly upon adrenergic stimulation of myocardium by cAMP-dependent protein kinase, and together with the phosphorylation of troponin-I and phospholamban contributes to the positive inotropy that results from adrenergic stimulation of the heart. Cardiac myosin binding protein C is phosphorylated by cAMP-dependent protein kinase on three sites in a myosin binding protein C specific N-terminal domain which binds to myosin-S2. This interaction with myosin close to the motor domain is likely to mediate the regulatory function of the protein. Cardiac myosin binding protein C is a common target gene of familial hypertrophic cardiomyopathy and most mutations encode N-terminal subfragments of myosin binding protein C. The understanding of the signalling interactions of the N-terminal region is therefore important for understanding the pathophysiology of myosin binding protein C associated cardiomyopathy. We demonstrate here by cosedimentation assays and isothermal titration calorimetry that the myosin-S2 binding properties of the myosin binding protein C motif are abolished by cAMP-dependent protein kinase-mediated tris-phosphorylation, decreasing the S2 affinity from a Kd of approximately 5 microM to undetectable levels. We show that the slow and fast skeletal muscle isoforms are no cAMP-dependent protein kinase substrates and that the S2 interaction of these myosin binding protein C isoforms is therefore constitutively on. The regulation of cardiac contractility by myosin binding protein C therefore appears to be a 'brake-off' mechanism that will free a specific subset of myosin heads from sterical constraints imposed by the binding to the myosin binding protein C motif.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Miocardio/metabolismo , Subfragmentos de Miosina/metabolismo , Calorimetría , Centrifugación , Fosforilación , Unión Proteica , Isoformas de ProteínasRESUMEN
We have recently suggested that similarly folded titin modules located at different sarcomeric regions have distinct molecular properties and stability. Could our selection of module boundaries have potentially influenced our conclusions? To address this question we expressed amino-terminally extended versions of the same modules and determined, with the use of CD and Fluorescence techniques, key thermodynamic parameters characterizing their stability. We present here our results which confirm our previous observations and show that, while amino-terminal extension has a profound effect on the stability of individual modules, it does not affect at all their folding pattern or their relative stabilities. Moreover, our data suggest that the selection of module boundaries can be of critical importance for the structural analysis of modular proteins in general, especially when a well-defined intron-exon topography is absent and proteolytic methods are inconclusive.
Asunto(s)
Proteínas Musculares/química , Oligopéptidos/fisiología , Pliegue de Proteína , Proteínas Quinasas , Secuencia de Aminoácidos , Dicroismo Circular , Conectina , Secuencia Conservada , Concentración de Iones de Hidrógeno , Inmunoglobulinas/química , Datos de Secuencia Molecular , Oligopéptidos/química , Desnaturalización Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , TermodinámicaRESUMEN
The giant muscle protein titin/connectin plays a crucial role in myofibrillogenesis as a molecular ruler for sarcomeric protein sorting. We describe here that the N-terminal titin immunoglobulin domains Z1 and Z2 interact specifically with telethonin in yeast two-hybrid analysis and protein binding assays. Immunofluorescence with antibodies against the N-terminal region of titin and telethonin detects both proteins at the Z-disc of human myotubes. Longer titin fragments, comprising a serine-proline-rich phosphorylation site and the next domain, do not interact. The interaction of telethonin with titin is therefore conformation-dependent, reflecting a possible phosphorylation regulation during myofibrillogenesis.