Conformation-regulated mechanosensory control via titin domains in cardiac muscle

The giant filamentous protein titin is ideally positioned in the muscle sarcomere to sense mechanical stimuli and transform them into biochemical signals, such as those triggering cardiac hypertrophy. In this review, we ponder the evidence for signaling hotspots along the titin filament involved in mechanosensory control mechanisms. On the way, we distinguish between stress and strain as triggers of mechanical signaling events at the cardiac sarcomere. Whereas the Z-disk and M-band regions of titin may be prominently involved in sensing mechanical stress, signaling hotspots within the elastic I-band titin segment may respond primarily to mechanical strain. Common to both stress and strain sensor elements is their regulation by conformational changes in protein domains

SH3 in muscles: Solution structure of the SH3 domain from nebulin

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

Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle

Kulke M, Neagoe C, Kolmerer B, et al. Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle. JOURNAL OF CELL BIOLOGY. 2001;154(5):1045-1058.Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and a-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-1-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu -calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms

SH3 in muscles: Solution structure of the SH3 domain from nebulin

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

Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin

Abstract BACKGROUND: Lesions in the gene for frataxin, a nuclear-encoded mitochondrial protein, cause the recessively inherited condition Friedreich's ataxia. It is thought that the condition arises from disregulation of mitochondrial iron homeostasis, with concomitant oxidative damage leading to neuronal death. Very little is, as yet, known about the biochemical function of frataxin. RESULTS: Here, we show that the mature form of recombinant frataxin behaves in solution as a monodisperse species that is composed of a 15-residue-long unstructured N terminus and an evolutionarily conserved C-terminal region that is able to fold independently. The structure of the C-terminal domain consists of a stable seven-stranded antiparallel beta sheet packing against a pair of parallel helices. The structure is compact with neither grooves nor cavities, features that are typical of iron-binding modules. Exposed evolutionarily conserved residues cover a broad area and all cluster on the beta-sheet face of the structure, suggesting that this is a functionally important surface. The effect of two clinically occurring mutations on the fold was checked experimentally. When the mature protein was titrated with iron, no tendency to iron-binding or to aggregation was observed. CONCLUSIONS: Knowledge of the frataxin structure provides important guidelines as to the nature of the frataxin binding partner. The absence of all the features expected for an iron-binding activity, the large conserved area on its surface and lack of evidence for iron-binding activity strongly support an indirect involvement of frataxin in iron metabolism. The effects of point mutations associated with Friedreich's ataxia can be rationalised by knowledge of the structure and suggest possible models for the occurrence of the disease in compound heterozygous patients

Towards a structural understanding of Friedreich's ataxia: the solution structure of frataxin

BACKGROUND: Lesions in the gene for frataxin, a nuclear-encoded mitochondrial protein, cause the recessively inherited condition Friedreich's ataxia. It is thought that the condition arises from disregulation of mitochondrial iron homeostasis, with concomitant oxidative damage leading to neuronal death. Very little is, as yet, known about the biochemical function of frataxin. RESULTS: Here, we show that the mature form of recombinant frataxin behaves in solution as a monodisperse species that is composed of a 15-residue-long unstructured N terminus and an evolutionarily conserved C-terminal region that is able to fold independently. The structure of the C-terminal domain consists of a stable seven-stranded antiparallel beta sheet packing against a pair of parallel helices. The structure is compact with neither grooves nor cavities, features that are typical of iron-binding modules. Exposed evolutionarily conserved residues cover a broad area and all cluster on the beta-sheet face of the structure, suggesting that this is a functionally important surface. The effect of two clinically occurring mutations on the fold was checked experimentally. When the mature protein was titrated with iron, no tendency to iron-binding or to aggregation was observed. CONCLUSIONS: Knowledge of the frataxin structure provides important guidelines as to the nature of the frataxin binding partner. The absence of all the features expected for an iron-binding activity, the large conserved area on its surface and lack of evidence for iron-binding activity strongly support an indirect involvement of frataxin in iron metabolism. The effects of point mutations associated with Friedreich's ataxia can be rationalised by knowledge of the structure and suggest possible models for the occurrence of the disease in compound heterozygous patients

Tissue-specific expression and α-actinin binding properties of the Z-disc titin: implications for the nature of vertebrate Z-discs

Titins are giant filamentous proteins which connect Z-discs and M-lines in the sarcomeres of vertebrate striated muscles. Comparison of the N-terminal region of titin (Z-disc region) from different skeletal and cardiac muscles reveals a 900-residue segment which is expressed in different length variants, dependent on tissue type. When searching for ligands of this differentially expressed domain by a yeast-two hybrid approach, we detected binding to alpha-actinin. The isolated alpha-actinin cDNAs were derived from the C-terminal region of the alpha-actinin isoform (alpha-actinin-2) encoded by the ACTN2 gene. Therefore, the two antiparallel subunits of an alpha-actinin-2 homodimer will attach to actin at their respective C termini, whereas they will bind to the Z-disc titin at their N termini. This may thus explain how alpha-actinins can cross-link antiparallel titin and thin filaments from opposing sarcomeres. The alpha-actinin-2 binding site of the Z-disc titin is located within a sequence of 45-residue repeats, referred to as Z-repeat region. Both the N-terminal and C-terminal Z-repeats have alpha-actinin binding properties and are expressed in all striated muscles. By contrast, the more central Z-repeats are expressed in slow and fast skeletal muscles, as well as embryonic and adult cardiac muscles, in different copy numbers. Such alternative splicing of the Z-disc titin appears to be important for the tissue and fibre type diversity of the Z-disc lattice