Structural predictions for the central domain of dystrophin

The amino acid sequence of dystrophin indicates that the molecule has globular N- and C-terminal domains separated by a long central rod domain. The central rod contains multiple repeats, about 100 amino acids long and of variable length. These diverge sufficiently in sequence that, in previous studies, only 14 of the most similar repeats have been aligned and analysed in any detail. We show here that a heptad pattern of hydrophobic residues is preserved across all repeats. Using the heptad pattern together with a consensus sequence template, we identified and aligned 25 repeats in the dystrophin rod sequence. Each repeat consists of a constant-length core helix of 54 residues, coupled via a short linker to a weakly conserved variable-length helix, and then via a second linker to the next core. The variable-length helix appears truncated in repeats 10 and 13 and extended in repeats 4 and 20. The extension of repeat 20 is particularly interesting since it corresponds to a hotspot of dystrophy-inducing mutations. Detailed modelling suggests that the classical Speicher-Marchesi [(1984) Nature 311, 177-180] model for spectrin may not be appropriate to dystrophin without some modification. We propose that whilst the repeating structural motif in dystrophin is probably a bead of triple coiled coil, this bead is twice as massive as, and out of phase with, those proposed for spectrin. Our model raises the possibility that the rod domain of dystrophin may confer elasticity on the molecule. Deletions which truncate this region would then reduce the extensibility of the molecule without affecting actin crosslinking, consistent with their typically producing the relatively benign Becker phenotype of muscular dystrophy.     

The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein

The complete sequence of the human Duchenne muscular dystrophy (DMD) cDNA has been determined. The 3685 encoded amino acids of the protein product, dystrophin, can be separated into four domains. The 240 amino acid N-terminal domain has been shown to be conserved with the actin-binding domain of alpha-actinin. A large second domain is predicted to be rod-shaped and formed by the succession of 25 triple-helical segments similar to the repeat domains of spectrin. The repeat segment is followed by a cysteine-rich segment that is similar in part to the entire COOH domain of the Dictyostelium alpha-actinin, while the 420 amino acid C-terminal domain of dystrophin does not show any similarity to previously reported proteins. The functional significance of some of the domains is addressed relative to the phenotypic characteristics of some Becker muscular dystrophy patients. Dystrophin shares many features with the cytoskeletal protein spectrin and alpha-actinin and is a large structural protein that is likely to adopt a rod shape about 150 nm in length.     

Sub-domains of the dystrophin rod domain display contrasting lipid-binding and stability properties

Dystrophin is a muscle scaffolding protein that establishes a structural link between the cytoskeleton and the extracellular matrix. Despite the large body of knowledge about the dystrophin gene and its interactions, the functional importance of the large central rod domain remains highly controversial. It is composed of 24 spectrin-like repeats interrupted by four hinges that delineate three sub-domains. We express repeat 1-3 and repeat 20-24 sub-domains, delineated by hinges 1-2 and 3-4 and the single repeats 2 and 23. We determine their lipid-binding properties, thermal and urea stabilities and refolding velocities. By using intrinsic tryptophan fluorescence spectroscopy and size exclusion chromatography, we show that repeat 2 and the repeat 1-3 sub-domain strongly interact with anionic lipids. By contrast, repeat 23 and the repeat 20-24 sub-domain do not interact with lipids. In addition, the repeat 1-3 sub-domain and repeat 2 are dramatically less stable and refold faster than the repeat 20-24 sub-domain and repeat 23. The contrasting properties of the two sub-domains clearly indicate that they make up two units of the rod domain that are not structurally interchangeable, thus providing molecular evidence supporting the observations on the biological function of dystrophin.     

Novel mutation in spectrin-like repeat 1 of dystrophin central domain causes protein misfolding and mild Becker muscular dystrophy

Mutations in the dystrophin gene without disruption of the reading frame often lead to Becker muscular dystrophy, but a genotype/phenotype correlation is difficult to establish. Amino acid substitutions may disrupt binding capacities of dystrophin and have a major impact on the functionality of this protein. We have identified two brothers (ages 8 and 10 years) with very mild proximal weakness, recurrent abdominal pain, and moderately elevated serum creatine kinase levels. Gene sequencing revealed a novel mutation in exon 11 of the dystrophin gene (c.1280T>C) leading to a L427P amino acid substitution in repeat 1 of the central rod domain. Immunostaining of skeletal muscle showed weak staining of the dystrophin region encoded by exons 7 and 8 corresponding to the end of the actin-binding domain 1 and the N-terminal part of hinge 1. Spectrofluorescence and circular dichroism analysis of the domain repeat 1-2 (R1-2) revealed partial misfolding of the L427P mutated protein as well as a reduced refolding rate after denaturation. Based on computational homology models of the wild-type and mutated R1-2, a molecular dynamics study showed an alteration in the flexibility of the structure, which also strongly affects the conformational space available in the N-terminal region of the fragment. Our results suggest that this missense mutation hinders the dynamic properties of the entire N-terminal region of dystrophin.     

Flexibility in the N‐terminal actin‐binding domain: Clues from in silico mutations and molecular dynamics

Dystrophin is a long, rod‐shaped cytoskeleton protein implicated in muscular dystrophy (MDys). Utrophin is the closest autosomal homolog of dystrophin. Both proteins have N‐terminal actin‐binding domain (N‐ABD), a central rod domain and C‐terminal region. N‐ABD, composed of two calponin homology (CH) subdomains joined by a helical linker, harbors a few disease causing missense mutations. Although the two proteins share considerable homology (>72%) in N‐ABD, recent structural and biochemical studies have shown that there are significant differences (including stability, mode of actin‐binding) and their functions are not completely interchangeable. In this investigation, we have used extensive molecular dynamics simulations to understand the differences and the similarities of these two proteins, along with another actin‐binding protein, fimbrin. In silico mutations were performed to identify two key residues that might be responsible for the dynamical difference between the molecules. Simulation points to the inherent flexibility of the linker region, which adapts different conformations in the wild type dystrophin. Mutations T220V and G130D in dystrophin constrain the flexibility of the central helical region, while in the two known disease‐causing mutants, K18N and L54R, the helicity of the region is compromised. Phylogenetic tree and sequence analysis revealed that dystrophin and utrophin genes have probably originated from the same ancestor. The investigation would provide insight into the functional diversity of two closely related proteins and fimbrin, and contribute to our understanding of the mechanism of MDys. Proteins 2015; 83:696–710. © 2015 Wiley Periodicals, Inc.     

The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries

Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.     

Molecular extensibility of mini-dystrophins and a dystrophin rod construct

Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophin's large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Delta2331 consists of repeats (R) and hinges (H) H1-R1-2 approximately H3 approximately R22-24-H4 linked by native (-) and non-native (approximately) sequence. This is compared to its core fragment R2 approximately H3 approximately R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23-42 degrees C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied betaI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.     

Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility

Most of dystrophin, the protein product of the Duchenne muscular dystrophy locus, is composed of spectrin-like repeats, suggesting that dystrophin is an elongated cytoskeletal molecule (Davison, M. D., and Critchley, D. R. (1988) Cell 52, 159-160; Koenig, M., Monaco, A. P., and Kunkel, L. M. (1988) Cell 53, 219-228). We present here a detailed analysis of the repeat domain of human dystrophin and propose that it is composed of 24 rather than 26 repeat units as previously suggested. Moreover, spacer sequences which do not align with the repeat consensus are present at the beginning and at the end of the repeat domain. Two other non-repeat spacers are found between repeat elements 3 and 4 and 19 and 20. The high proline content of each spacer suggests that it might represent a hinge. Using five new anti-dystrophin antisera and two previously described antisera (Hoffman, E. P., Brown, R. H., Jr., and Kunkel, L. M. (1987a) Cell 51, 919-928) to detect different dystrophin peptides after proteolytic cleavage, we show that the four hinge segments are sensitive sites for proteolysis. We present a model for a membrane-associated network of dystrophin in which the hinges play a key role by conferring flexibility to the network and thus resilience to the membrane.     

Molecular diagnosis of Duchenne/Becker muscular dystrophy by polymerase chain reaction and microsatellite analysis

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive genetic disorders resulting from mutations in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central region of the gene. The remaining DMD/BMD cases show no deletions, so they cannot be easily identified by current strategies. In these DMD/BMD families, a linkage analysis that involves DNA markers of the flanking and intragenic dystrophin gene are necessary for carrier and prenatal diagnosis. We analyzed eighteen deletion-prone exons of the gene by a polymerase chain reaction (PCR) in order to characterize the molecular defects of the dystrophin gene in Korean DMD/BMD families. We also performed a linkage analysis to assess the usefulness and application of six short tandem repeat markers for molecular diagnosis in the families. We observed a deletion that eliminated the exon 50. Also, a linkage analysis in the families with six short tandem repeat (STR) markers showed heterozygosity at most of the STR markers. The haplotype analysis was useful for detecting the carrier status. This study will be helpful for a molecular diagnosis of DMD/BMD families in the Korean population.     

Transgenic mdx mice expressing dystrophin with a deletion in the actin- binding domain display a "mild Becker" phenotype

The functional significance of the actin-binding domain of dystrophin, the protein lacking in patients with Duchenne muscular dystrophy, has remained elusive. Patients with deletions of this domain (domain I) typically express low levels of the truncated protein. Whether the moderate to severe phenotypes associated with such deletions result from loss of an essential function, or from reduced levels of a functional protein, is unclear. To address this question, we have generated transgenic mice that express wild-type levels of a dystrophin deleted for the majority of the actin-binding domain. The transgene derived protein lacks amino acids 45-273, removing 2 of 3 in vitro identified actin interacting sites and part of hinge 1. Examination of the effect of this deletion in mice lacking wild-type dystrophin (mdx) suggests that a functional domain I is not essential for prevention of a dystrophic phenotype. However, in contrast to deletions in the central rod domain and to full-length dystrophin, both of which are functional at only 20% of wild-type levels, proteins with a deletion in domain I must be expressed at high levels to prevent a severe dystrophy. These results are also in contrast to the severe dystrophy resulting from truncation of the COOH-terminal domain that links dystrophin to the extracellular matrix. The mild phenotype observed in mice with domain I-deletions indicates that an intact actin-binding domain is not essential, although it does contribute to an important function of dystrophin. These studies also suggest the link between dystrophin and the subsarcolemmal cytoskeleton involves more than a simple attachment of domain I to actin filaments.     

Rapid detection of CA polymorphisms in cloned DNA: application to the 5'' region of the dystrophin gene.

To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.     

Tandemly repeating peptide motifs and their secondary structure in Ceratitis capitata eggshell proteins Ccs36 and Ccs38.

Evidence from amino acid composition, Fourier transform analysis of primary structure and secondary structure prediction suggests a tripartite structure for Ceratitis capitata eggshell proteins Ccs36 and Ccs38, which consists of a central domain and two flanking 'arms'. The proteins, apparently, contain tandemly repeating peptide motifs specific for each domain of the tripartite structure. The central domain of both proteins, which exhibits extensive sequence homology with the corresponding domains of Drosophila melanogaster proteins s36 and s38, is formed by tandem repeats of an octapeptide-X-X-X-Z-Z-Z-Z-Z- (where X = large hydrophobic residue and Z = beta-turn former residue) and its variants. It is predicted to adopt a compact, most probably twisted, antiparallel beta-pleated sheet structure of beta-sheet strands regularly alternating with beta-turns or loops. The central domains of Ccs36 and Ccs38 share structural similarities, but they are recognizably different. The 'arms' of the proteins presumably serving for protein and species-specific functions differ substantially from those of Drosophila melanogaster. In Ccs36, the C-terminal 'arm' is formed by, almost precise, tandem repeats of an octapeptide-Y-X-A-A-P-A-A-S- (X = G or S), whereas the N-terminal 'arm' contains repeats of the octapeptide -Z-Z-Z-A-X-A-A-Z- (X = Q, N or E and Z a beta-turn former). In both 'arms' alpha-helices are predicted, alternating with beta-turns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic analysis of human chromosome 10q and 4q telomeres suggests a common origin

The subtelomeric region of human chromosome 4q contains the locus for facioscapulohumeral muscular dystrophy (FSHD). The FSHD mutation is a deletion within an array of 3.3-kb tandem repeats (D4Z4). The disease mechanism is unknown but is postulated to involve position effect. A closely related 3.3-kb array on chromosome 10qter, in contrast, is not associated with a disease phenotype. We show here that the 4q homology on chromosome 10 is not confined to the 3.3-kb repeats but extends both proximally (42 kb) and distally to include the telomere. We have also identified the most distal expressed gene on 10q known so far, mapping only 96 kb from the 3.3-kb repeat array. A 4q variant has also been identified; there is 92%nucleotide identity between the two 4q forms, 4qA and 4qB. The 4qter and 10qter forms show homology to other chromosome ends, including 4p, 21q, and 22q, and these regions may represent a relatively common subtelomeric domain.     

A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.     

Mild deficiency of dystrophin-associated proteins in Becker muscular dystrophy patients having in-frame deletions in the rod domain of dystrophin.

The dystrophin-glycoprotein complex spans the sarcolemma to provide a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in skeletal muscle. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins in the sarcolemma, thus causing the disruption of the dystrophin-glycoprotein complex and the loss of the linkage to the extracellular matrix. The resulting sarcolemmal instability is presumed to render muscle fibers susceptible to necrosis. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from patients with Becker muscular dystrophy (BMD), a milder allelic form of DMD. BMD patients having in-frame deletions in the rod domain of dystrophin showed a mild to moderate reduction in all of the dystrophin-associated proteins in the sarcolemma, but this reduction was not as severe as that in DMD patients. The reduction of the immunostaining for the dystrophin-associated proteins showed a good correlation with that for dystrophin in both intensity and distribution. Our results indicate that (1) the abnormality of the sarcolemmal glycoprotein complex, which is similar to but milder than that in DMD patients, also exists in these BMD patients and (2) the rod domain of dystrophin is not crucial for the interaction with the dystrophin-associated proteins.     

Duchenne muscular dystrophy and dystrophin: Sequence homology observations

Duchenne muscular dystrophy (DMD) is a genetically transmitted disease characterized by progressive muscle weakness and usually leads to death. DMD results from the absence, deficiency or dysfunction of the protein dystrophin. Analysis of protein data bases, including homology alignments and domain recognition patterns, have located highly significant correlations between dystrophin and other calcium regulating proteins. In particular, a major portion of the dystrophin sequence has been found to contain repeating units of approximately 100 amino acid residues. These repeating units were found to exhibit significant homology to troponin I. Troponin I has been found to bind to the calcium binding proteins calmodulin and troponin C. The regions of highest homology were characterized by patterns of high localization of charged amino acids and thus could represent a possible calmodulin or troponin C surface accessible binding site. Since subcellular localization studies have indicated that dystrophin is associated with the triadic junction, these findings imply that dystrophin could be involved in controlling intracellular calcium homeostasis.Special issue dedicated to Dr. Lawrence Austin.     

Homology of the precursor of pulmonary surfactant-associated protein SP-B with prosaposin and sulfated glycoprotein 1.

The precursor of pulmonary surfactant-associated protein, SP-B, is composed of an NH2-terminal domain of 30 residues (a-type domain) and three tandem repeats of about 90 residues (b-type domain); biophysically active mature SP-B corresponds to the second b-type repeat. Consensus sequences constructed for the b-type repeats were used to search the data base for homologous sequences, and the search has revealed that prosaposin and sulfated glycoprotein 1 show a remarkable homology with these repeats. The domain organizations of the latter proteins, however, differ from that of SP-B precursor inasmuch as they contain four tandem copies of the b-type domain and a-type domains are present both in the NH2-terminal and COOH-terminal parts of the proteins. The implications of the homology of saposins and SP-B for their structure and function are discussed.     

The structure of a tandem pair of spectrin repeats of plectin reveals a modular organization of the plakin domain

Plectin is a large and versatile cytoskeletal linker and member of the plakin protein family. Plakins share a conserved region called the plakin domain located near their N terminus. We have determined the crystal structure of an N-terminal fragment of the plakin domain of plectin to 2.05 A resolution. This region is adjacent to the actin-binding domain and is required for efficient binding to the integrin alpha6beta4 in hemidesmosomes. The structure is formed by two spectrin repeats connected by an alpha-helix that spans these two repeats. While the first repeat is very similar to other known structures, the second repeat is structurally different with a hydrophobic core, narrower than that in canonical spectrin repeats. Sequence analysis of the plakin domain revealed the presence of up to nine consecutive spectrin repeats organized in an array of tandem modules, and a Src-homology 3 domain inserted in the central spectrin repeat. The structure of the plakin domain is reminiscent of the modular organization of members of the spectrin family. The architecture of the plakin domain suggests that it forms an elongated and flexible structure, and provides a novel molecular explanation for the contribution of plectin and other plakins to the elasticity and stability of tissues subjected to mechanical stress, such as the skin and striated muscle.