The WW domain of dystrophin requires EF-hands region to interact with beta-dystroglycan.

Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein beta-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with beta-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of beta-dystroglycan. This region of dystrophin implicated in binding beta-dystroglycan contains four modular protein domains: a WW domain, two putative Ca2+-binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38-40 amino acids with two highly conserved tryptophan residues spaced 20-22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding beta-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of beta-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxy-terminus of beta-dystroglycan occurs via a beta-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/beta-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.     

Structure of a WW domain containing fragment of dystrophin in complex with beta-dystroglycan

Dystrophin and beta-dystroglycan are components of the dystrophin-glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of beta-dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of beta-dystroglycan. Here we report the crystal structure of this portion of dystrophin in complex with the proline-rich binding site in beta-dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The beta-dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains.     

Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex

The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein-protein interaction. These domains (WW, EF hands, ZZ and H1-H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxy-terminus. We found that none of the carboxy-terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage-displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in beta-dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW-mediated binding to PPxY peptides and to beta-dystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin beta-dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca(2+)-dependent binding of calmodulin. The remaining two motifs found in the carboxy-terminus (H1-H2) mediate the formation of utrophin-dystrobrevin hybrids but do not select ligands in a repertoire of random nonapeptides.     

Mammalian alpha 1- and beta 1-syntrophin bind to the alternative splice- prone region of the dystrophin COOH terminus

The carboxy-terminal region of dystrophin has been suggested to be crucially important for its function to prevent muscle degeneration. We have previously shown that this region is the locus that interacts with the sarcolemmal glycoprotein complex, which mediates membrane anchoring of dystrophin, as well as with the cytoplasmic peripheral membrane protein, A0 and beta 1-syntrophin (Suzuki, A., M. Yoshida, K. Hayashi, Y. Mizuno, Y. Hagiwara, and E. Ozawa. 1994. Eur. J. Biochem. 220:283- 292). In this work, by using the overlay assay technique developed previously, we further analyzed the dystrophin-syntrophin/A0 interaction. Two forms of mammalian syntrophin, alpha 1- and beta 1- syntrophin, were found to bind to very close but discrete regions on the dystrophin molecule. Their binding sites are located at the vicinity of the glycoprotein-binding site, and correspond to the amino acid residues encoded by exons 73-74 which are alternatively spliced out in some isoforms. This suggests that the function of syntrophin is tightly linked to the functional diversity among dystrophin isoforms. Pathologically, it is important that the binding site for alpha 1- syntrophin, which is predominantly expressed in skeletal muscle, coincides with the region whose deletion was suggested to result in a severe phenotype. In addition, A0, a minor component of dystrophin- associated proteins with a molecular mass of 94 kD which is immunochemically related to syntrophin, binds to the same site as beta 1-syntrophin. Finally, based on our accumulated evidence, we propose a revised model of the domain organization of dystrophin from the view point of protein-protein interactions.     

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.     

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.     

Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain

Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex.     

The structure of the N-terminal actin-binding domain of human dystrophin and how mutations in this domain may cause Duchenne or Becker muscular dystrophy

BACKGROUND: Dystrophin is an essential component of skeletal muscle cells. Its N-terminal domain binds to F-actin and its C terminus binds to the dystrophin-associated glycoprotein (DAG) complex in the membrane. Dystrophin is therefore thought to serve as a link from the actin-based cytoskeleton of the muscle cell through the plasma membrane to the extracellular matrix. Pathogenic mutations in dystrophin result in Duchenne or Becker muscular dystrophy. RESULTS: The crystal structure of the dystrophin actin-binding domain (ABD) has been determined at 2.6 A resolution. The structure is an antiparallel dimer of two ABDs each comprising two calponin homology domains (CH1 and CH2) that are linked by a central alpha helix. The CH domains are both alpha-helical globular folds. Comparisons with the structures of utrophin and fimbrin ABDs reveal that the conformations of the individual CH domains are very similar to those of dystrophin but that the arrangement of the two CH domains within the ABD is altered. The dystrophin dimer reveals a change of 72 degrees in the orientation of one pair of CH1 and CH2 domains (from different monomers) relative to the other pair when compared with the utrophin dimer. The dystrophin monomer is more elongated than the fimbrin ABD. CONCLUSIONS: The dystrophin ABD structure reveals a previously uncharacterised arrangement of the CH domains within the ABD. This observation has implications for the mechanism of actin binding by dystrophin and related proteins. Examining the position of three pathogenic missense mutations within the structure suggests that they exert their effects through misfolding of the ABD, rather than through disruption of the binding to F-actin.     

A new model for the interaction of dystrophin with F-actin

The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super- family of proteins. However, we failed to observe dystrophin- glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin- glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F- actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.     

The signal transducer gp130: solution structure of the carboxy-terminal domain of the cytokine receptor homology region

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.     

The carboxy-terminal domain of glycoprotein N of human cytomegalovirus is required for virion morphogenesis

Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.     

A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region

The E1 glycoprotein of the avian coronavirus infectious bronchitis virus contains a short, glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxy-terminal cytoplasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in infected cells. E1 proteins with precise deletions of the first and second or the second and third membrane-spanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain can function as an internal signal sequence. The mutant protein with only the first transmembrane domain accumulated intracellularly like the wild-type protein, but the mutant protein with only the third transmembrane domain was transported to the cell surface. This result suggests that information specifying accumulation in the Golgi region resides in the first transmembrane domain, and provides the first example of an intracellular membrane protein that is transported to the plasma membrane after deletion of a specific domain.     

Utrophin lacks the rod domain actin binding activity of dystrophin

We previously identified a cluster of basic spectrin-like repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419-28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11-17) or the homologous region of the utrophin rod domain (UTROR11-16). Although UTROR11-16 is 64% similar and 41% identical to DYSR11-17, UTROR11-16 (pI = 4. 86) lacks the basic character of the repeats found in DYSR11-17 (pI = 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly alpha-helical fold and existed as a highly soluble monomer. DYSR11-17 bound F-actin with an apparent K(d) of 7.3 +/- 1.3 microM and a molar stoichiometry of 1:5. Significantly, UTROR11-16 failed to bind F-actin at concentrations as high as 100 microM. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin.     

ZZ domain of dystrophin and utrophin: topology and mapping of a beta-dystroglycan interaction site

Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor dystroglycan. Dystrophin and its autosomal homologue utrophin interact with beta-dystroglycan via their highly conserved C-terminal cysteine-rich regions, comprising the WW domain (protein-protein interaction domain containing two conserved tryptophan residues), EF hand and ZZ domains. The EF hand region stabilizes the WW domain providing the main interaction site between dystrophin or utrophin and dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilizes the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and beta-dystroglycan. Using bacterially expressed ZZ domain, we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc-binding regions within the ZZ domain by SPOTs overlay assays. Epitope mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with beta-dystroglycan. The epitope was localized to the conformationally sensitive second zinc-binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin form a crucial part of the contact region between dystrophin and beta-dystroglycan and provide new insight into ZZ domain organization and function.     

Shedding of vesicular stomatitis virus soluble glycoprotein by removal of carboxy-terminal peptide.

A comparison of partial NH2-terminal sequences of vesicular stomatitis viral glycoprotein G (molecular weight, 69,000) and the soluble extracellular glycoprotein antigen Gs (molecular weight, 57,000) shows that both of the sequences are identical. Tryptic fingerprint analyses show that Gs lacks the carboxy-terminal region containing the membrane-anchoring hydrophobic domain of G. These results suggest that Gs is formed by cleavage in the carboxy-terminal region of G.     

Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface.

Polyclonal and monoclonal antibodies were raised against a synthetic peptide containing the 15 carboxy-terminal amino acids (497-511) of vesicular stomatitis virus glycoprotein (VSV-G). The polyclonal antibodies (alpha P4) reacted with epitopes distributed along the 15-residue peptide, whereas the monoclonal antibody (P5D4) reacted with one epitope containing the five carboxy-terminal amino acids. Both types of antibodies recognized the cytoplasmic domain of VSV-G synthesized by tissue culture cells infected with the temperature-sensitive 045-VSV mutant (ts045-VSV). They recognized immature forms of VSV-G in the rough endoplasmic reticulum (RER) and Golgi complex, as well as mature VSV-G at the cell surface and in budding virus. The effect of these antibodies on intracellular transport and maturation of VSV-G was studied by microinjection. Both divalent antibodies (alpha P4 and P5D4) blocked transport of VSV-G to the cell surface. Monovalent Fab' fragments of alpha P4 (alpha P4-Fabs) also interfered with the appearance of VSV-G at the cell surface; Fab fragments of P5D4 (P5D4-Fabs), however, had no inhibitory effect. These results suggest that accessibility of a cytoplasmic domain, located within the sequence of amino acids 497-506 of the carboxy-terminal tail, is essential for transport of VSV-G to the cell surface.     

Caveolin-3 directly interacts with the C-terminal tail of beta -dystroglycan. Identification of a central WW-like domain within caveolin family members

Caveolin-3, the most recently recognized member of the caveolin gene family, is muscle-specific and is found in both cardiac and skeletal muscle, as well as smooth muscle cells. Several independent lines of evidence indicate that caveolin-3 is localized to the sarcolemma, where it associates with the dystrophin-glycoprotein complex. However, it remains unknown which component of the dystrophin complex interacts with caveolin-3. Here, we demonstrate that caveolin-3 directly interacts with beta-dystroglycan, an integral membrane component of the dystrophin complex. Our results indicate that caveolin-3 co-localizes, co-fractionates, and co-immunoprecipitates with a fusion protein containing the cytoplasmic tail of beta-dystroglycan. In addition, we show that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of beta-dystroglycan that contains a PPXY motif. As the WW domain of dystrophin recognizes the same site within beta-dystroglycan, we also demonstrate that caveolin-3 can effectively block the interaction of dystrophin with beta-dystroglycan. In this regard, interaction of caveolin-3 with beta-dystroglycan may competitively regulate the recruitment of dystrophin to the sarcolemma. We discuss the possible implications of our findings in the context of Duchenne muscular dystrophy.     

The crystal structure of the carboxy-terminal domain of human translation initiation factor eIF5

The carboxy-terminal domain (CTD) of eukaryotic initiation factor 5 (eIF5) plays a central role in the formation of the multifactor complex (MFC), an important intermediate for the 43 S pre-initiation complex assembly. The IF5-CTD interacts directly with the translation initiation factors eIF1, eIF2-beta, and eIF3c, thus forming together with eIF2 bound Met-tRNA(i)(Met) the MFC. In this work we present the high resolution crystal structure of eIF5-CTD. This domain of the protein is exclusively composed out of alpha-helices and is homologous to the carboxy-terminal domain of eIF2B-epsilon (eIF2Bepsilon-CTD). The most striking difference in the two structures is an additional carboxy-terminal helix in eIF5. The binding sites of eIF2-beta, eIF3 and eIF1 were mapped onto the structure. eIF2-beta and eIF3 bind to non-overlapping patches of negative and positive electrostatic potential, respectively.