The membrane-cytoskeleton interface: the role of dystrophin and utrophin

Recent studies with transgenic animals have considerably advanced our knowledge of the roles of dystrophin and utrophin in both muscle and non-muscle tissues. Rigorous analyses of the roles of the various mdx mutations in mice, as well as the use of artificial transgenes in an mdx background, are beginning to define the functional importance of various regions of the dystrophin protein in normal muscle. Furthermore, recent biochemical analyses have revealed new insights into the role and organization of dystrophin at the membrane-cytoskeleton interface. Transgenic approaches have also revealed surprising and encouraging results with respect to utrophin. Against expectations, the long-awaited utrophin knockout mice have a remarkably mild phenotype with only subtle changes in neuromuscular junction architecture. On the other hand, mdx mice transgenic for a mini-utrophin construct showed rescue of the muscular dystrophy phenotype, clearly an encouraging finding with obvious therapeutic possibilities. These and other recent findings are discussed in the context of the structure and function of dystrophin and utrophin at the membrane-cytoskeleton interface.     

Crystal structure of the actin-binding protein actophorin from Acanthamoeba

Tertiary butyl alcohol and trichloroacetic acid are known to be contaminants in drinking water. In order to evaluate the interactive toxicity of t-butyl alcohol with trichloroacetic acid, young male Wistar rats were dosed through water at a dose level of t-butyl alcohol (TBA)-0.5% (v/v), trichloroacetic acid (TCA)-25 ppm and a combined dose of TBA + TCA (0.5% v/v TBA-25 ppm TCA) for a period of 10 weeks ad libitum and were maintained on normal diet. The control animals received plain water and normal diet. The liver and kidney histology was undertaken to see whether subtoxic administration of TBA and TCA individually as well as combined administration for a period of 10 weeks would bring about any histological alterations. It was observed that TBA, TCA and TBA + TCA caused histological alterations in the liver such as centrilobular necrosis, vacuolation in hepatocytes and loss of hepatic architecture. TBA and TBA + TCA caused periportal proliferation and lymphocytic infiltration. Hypertrophy of hepatocytes in the periportal area was a characteristic feature in the liver of TCA treated rats. Moreover, in the histology of the kidney, in the three treated groups, degeneration of renal tubules, with syncitial arrangements of the nucleus of renal tubular epithelial cells was evident. In addition to this, degeneration of the basement membrane of the Bowmans capsule, diffused glomeruli and vacuolation of glomeruli was also evident in the three treated rat kidneys. Renal tubular proliferation in certain areas was also evident in certain areas of the kidney in TCA treated rats. The results indicate that, TBA and TCA do bring about alterations in histology of liver and kidney, but on combined administration, do not show enhanced toxicity in the form of increased hepatic and renal injury.     

Indications that the Cgamma2 homology region is not a regular domain

The Cgamma2 homology region of rabbit IgG does not behave like a domain. Thus, there is no trans-interaction between the two Cgamma2 regions; instead there is an unusual cis-interaction between Cgamma2 and Cgamma3 regions. The observations were made on the plasmin digestion products Facb (IgG minus the Cgamma3 region) and pFc' (Cgamma3 region), which did not dissociate under neutral conditions but dissociated in 3M guanidine solution (that is, cis-interaction between Cgamma2 and Cgamma3). The Facb fragment split into subunits with equal molecular weights under neutral conditions on partial reduction and alkylation (that is, lack of trans-interaction between the two Cgamma2 in the molecule).     

The structure of the membrane protein squalene-hopene cyclase at 2.0 A resolution

Squalene cyclases catalyze a cationic cyclization cascade, which is homologous to a key step in cholesterol biosynthesis. The structure of the enzyme from Alicyclobacillus acidocaldarius has been determined in a new crystal form at 2.0 A resolution (1 A=0.1 nm) and refined to an R-factor of 15.3 % (Rfree=18.7 %). The structure indicates how the initial protonation and the final deprotonation of squalene occur and how the transient carbocations are stabilized. The pathways of the flexible educt squalene from the membrane interior to the active center cavity and of the rigid fused-ring product hopene in the reverse direction are discussed. The enzyme contains eight so-called QW-sequence repeats that fortify the alpha/alpha-barrels by an intricate interaction network. They are unique to the known triterpene cyclases and are presumed to shield these enzymes against the released enthalpy of the highly exergonic catalyzed reaction. The enzyme is a monotopic membrane protein, the membrane-binding interactions of which are described and compared with those of two prostaglandin-H2 synthase isoenzymes, the only other structurally characterized proteins of this type. In the crystals the membrane-binding regions face each other, suggesting a micelle-type detergent structure between them.     

Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the pleckstrin homology domain from beta-spectrin

Human serum amyloid P component (SAP) is a normal plasma glycoprotein and the precursor of amyloid P component which is a universal constituent of the abnormal tissue deposits in amyloidosis. X-ray and neutron scattering data showed that pentameric or decameric ring structures for SAP in solution are readily distinguished. Further neutron data collection showed that SAP pentamers were reproducibly obtained in the presence of Ca2+ at pH 5.5 or in the presence of methyl 4,6-O-(1-carboxyethylidene)-beta-d-galactopyranoside (MObetaDG) and Ca2+ at pH 6.0 to 8.0, while SAP decamers were obtained in the presence of EDTA between pH 5.5 and 8.0. SAP pentamers have a mean X-ray RG of 3.99(+/-0.11) nm and a mean neutron RG of 3.69(+/-0.12) nm in 100% 2H2O. SAP decamers have a mean X-ray RG of 4.23(+/-0.12) nm and a mean neutron RG of 4.09(+/-0.14) nm in 100% 2H2O. The absorption coefficients of SAP pentamers and decamers differ by 10%. If we infer that the two alpha-helical A-faces are in contact with each other in the SAP decamer, the lack of structural change of the decamer with pH may be explained by the absence of His residues from the A-face of the SAP pentamer, and the change in absorption coefficients is compatible with the presence of Trp residues at this A-face. The rigid ring structure of pentameric SAP provided a test of scattering curves calculated from crystal structures. The only structural unknown is the orientation of the five chemically homogeneous oligosaccharide chains relative to the protein, but extended oligosaccharide structures were found to account for its scattering curve. X-ray scattering curves were best calculated using a hydrated structure, while neutron scattering curves were best calculated using an unhydrated structure. The outcome of these analyses was used to model the structure of decameric SAP. The evaluation of 640 structures for two SAP pentamers brought face-to-face to form SAP decamers gave better curve fits for structures in which the two A-faces were in contact with each other, in which it is likely that the two pentamers were out of alignment by a rotation of 36 degrees and the oligosaccharide chains were extended.     

Reduced sarcolemmal dystrophin distribution and upregulation of utrophin in the cardiac and skeletal muscles of CHF-146 dystrophic hamsters

Abnormalities in the dystrophic gene product, dystrophin, have been implicated in initiating the primary membrane defect and excessive intracellular calcium accumulation (EICA), which play fundamental pathogenic roles in hereditary muscular dystrophy (HMD). Two other cytoskeletal proteins, spectrin and utrophin, bear remarkable structural and functional homologies to dystrophin. CHF-146 strain dystrophic hamsters (DH), like patients with Duchenne muscular dystrophy (DMD), die prematurely from cardiopulmonary insufficiency, focal myonecrosis, and progressive degeneration of the cardiac and skeletal muscles with EICA. Although DH present a suitable model for HMD, there are controversies concerning their dystrophin and utrophin status. Using immunocytochemistry and Western blotting, we studied dystrophin, spectrin and utrophin anomalies in the cardiac and skeletal muscles of 6-mo-old male DH. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as controls. Sarcolemmal dystrophin staining was much weaker and interruptive in the DH. The densitometric analysis of the immunoblots revealed that dystrophin is reduced in DH by 83% in cardiac muscle (p < 0.0001), and by 50% in skeletal muscle (p < 0.0001). We conclude that sarcolemmal dystrophin distribution is markedly reduced and discontinuous in the cardiac and skeletal muscles of DH, with simultaneous upregulation of utrophin and a varied degree of spectrin labelling. This observation suggests that reduced sarcolemmal dystrophin is associated with membrane hyperpermeability, which leads to progressive muscle degeneration via EICA and segmental necrosis in DH. As in DMD, utrophin appears to play an important compensatory role in hamster dystrophinopathy.     

Structure and mutagenesis of the Dbl homology domain

Guanine nucleotide exchange factors in the Dbl family activate Rho GTPases by accelerating dissociation of bound GDP, promoting acquisition of the GTP-bound state. Dbl proteins possess a approximately 200 residue catalytic Dbl-homology (DH) domain, that is arranged in tandem with a C-terminal pleckstrin homology (PH) domain in nearly all cases. Here we report the solution structure of the DH domain of human PAK-interacting exchange protein (betaPIX). The domain is composed of 11 alpha-helices that form a flattened, elongated bundle. The structure explains a large body of mutagenesis data, which, along with sequence comparisons, identify the GTPase interaction site as a surface formed by three conserved helices near the center of one face of the domain. Proximity of the site to the DH C-terminus suggests a means by which PH-ligand interactions may be coupled to DH-GTPase interactions to regulate signaling through the Dbl proteins in vivo.     

The three-dimensional structure of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides refined at 2.0 A resolution

BACKGROUND: Glucose 6-phosphate dehydrogenase (G6PD) is the first enzyme of the pentose phosphate pathway. Normally the pathway is synthetic and NADP-dependent, but the Gram-positive bacterium Leuconostoc mesenteroides, which does not have a complete glycolytic pathway, also uses the oxidative enzymes of the pentose phosphate pathway for catabolic reactions, and selects either NAD or NADP depending on the demands for catabolic or anabolic metabolism. RESULTS: The structure of G6PD has been determined and refined to 2.0 A resolution. The enzyme is a dimer, each subunit consisting of two domains. The smaller domain is a classic dinucleotide-binding fold, while the larger one is a new beta+ alpha fold, not previously seen, with a predominantly antiparallel nine-stranded beta-sheet. There are significant structural differences in the coenzyme-binding domains of the two subunits, caused by Pro 149 which is cis in one subunit and trans in the other. CONCLUSIONS: The structure has allowed us to propose the location of the active site and the coenzyme-binding site, and suggests the role of many of the residues conserved between species. We propose that the conserved Arg46 would interact with both the adenine ring and the 2'-phosphate of NADP. Gln47, which is not conserved, may contribute to the change from NADP to dual coenzyme specificity. His178, in a nine-residue peptide conserved for all known sequences, binds a phosphate in the active site pocket. His240 is the most likely candidate for the base to oxidize the 1-hydroxyl group of the glucose 6-phosphate substrate.     

Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins

The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. We have identified a 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing and report here its crystal structure. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins.     

The gelatin-binding site of the second type-II domain of gelatinase A/MMP-2

We have shown previously that all three fibronectin type-II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to localize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent-induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild-type module. However, its gelatin affinity was severely impaired compared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PDC-109 places Y37 on the right-hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.     

A cluster of basic repeats in the dystrophin rod domain binds F-actin through an electrostatic interaction

The dystrophin rod domain is composed of 24 spectrin-like repeats and was thought to act mainly as a flexible spacer between the amino-terminal actin binding domain and carboxyl-terminal membrane-associated domains. We previously demonstrated that a fragment of the dystrophin rod domain also binds F-actin. However, the nature and extent of rod domain association with F-actin is presently unclear. To begin addressing these questions, we characterized two recombinant proteins representing adjacent regions of the dystrophin rod. DYS1416 (amino acids 1416-1880) bound F-actin with a Kd of 14.2 +/- 5.2 microM and a stoichiometry of 1 mol:mol of actin. However, DYS1030 (amino acids 1030-1494) failed to bind F-actin, suggesting that not all rod domain repeats are capable of binding F-actin. Interestingly, DYS1416 corresponds to a unique region of the dystrophin rod rich in basic amino acids, whereas DYS1030 is composed mainly of acidic repeats. This observation suggested that DYS1416 may interact with acidic actin filaments through an electrostatic interaction. Supporting this hypothesis, actin binding by DYS1416 was dramatically inhibited by increasing ionic strength. We suggest that electrostatic interactions between basic spectrin-like repeats and actin filaments may contribute to the actin binding activity of other members of the actin cross-linking protein family.     

Solution structure of the Eps15 homology domain of a human POB1 (partner of RalBP1)

The solution structure of the Eps15 homology (EH) domain of a human POB1 (partner of RaIBP1) has been determined by uniform 13C/15N labeling and heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The POB1 EH domain consists of two EF-hand structures, and the second one binds a calcium ion. In the calcium-bound state, the orientation of the fourth alpha-helix relative to the other helices of the POB1 EH domain is slightly different from that of calbindin, and much more different from those of calmodulin and troponin C, on the basis of their atomic coordinates.     

A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors

This paper describes the identification of a new family of mammalian genes that encode secreted proteins containing homology to the cysteine-rich ligand-binding domain found in the frizzled family of transmembrane receptors. The secreted frizzled-related proteins (sFRPs) are approximately 30 kDa in size, and each contains a putative signal sequence, a frizzled-like cysteine-rich domain, and a conserved hydrophilic carboxy-terminal domain. The sFRPs are not the products of differential splicing of the known frizzled genes. Glycosylphosphatidylinositol-anchored derivatives of sFRP-2 and sFRP-3 produced in transfected human embryonic kidney cells confer cell-surface binding by the Drosophila Wingless protein. These observations suggest that sFRPs may function in vivo to modulate Wnt signaling, or, alternatively, as novel ligands for as yet unidentified receptors.     

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin

A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3' end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.     

Consequences of the combined deficiency in dystrophin and utrophin on the mechanical properties and myosin composition of some limb and respiratory muscles of the mouse

The mechanical properties and the myosin isoform composition were studied in three isolated muscles (EDL, soleus, diaphragm) of mutant mice lacking both dystrophin and utrophin (dko). They were compared with the corresponding muscles of the normal and the dystrophin-deficient (mdx) and the utrophin-deficient (uko) mice. In comparison with mdx muscles, dko muscles show a significant reduction of the normalized isometric force, confirmed by the reduced muscular activity of the whole animal. Kinetics parameters (twitch time-to-peak and half-relaxation time) were slightly reduced, and the maximal speed of shortening of soleus, Vmax, was reduced by 30%. The maximal power output (muW/mm3) was reduced by 50% in dko soleus. In the three muscles studied, the relative myosin heavy chains (MHC) composition showed a shift towards slower isoforms. dko EDL presented a dramatic decrease of the resistance ot tetanic contraction with forced lengthenings (eccentric contractions), while muscle lacking only utrophin (uko mutants) display a normal resistance to this exacting mechanical challenge. These experiments suggest that lack of both dystrophin and utrophin is very detrimental to the mice and that mechanical properties of the muscles may explain the overall phenotype. Moreover these results bring some support to the idea that the expression of utrophin in mdx muscle compensates, to some extent, for the lack of dystrophin.     

Refined structure of dimeric diphtheria toxin at 2.0 A resolution

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.     

The pleckstrin homology domain is the principal link between the insulin receptor and IRS-1

Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1(PH) contains a pleckstrin homology (PH) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN was proposed to contain another functional PTB domain. Peptide competition experiments demonstrated that the IH2(PTB) in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1(PH) or the SAIN regions. In 32D cells the IH1(PH) was essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activity and p70(s6k) phosphorylation. In contrast, the IH2(PTB) and the SAIN regions were not required for these insulin actions; however, the IH2(PTB) improved the coupling between IRS-1 and the insulin receptor. Overexpression of the insulin receptor in 32DIR cells increased IRS-1 tyrosine phosphorylation and mediated insulin-stimulated DNA synthesis. The sensitivity of these responses was partially reduced by deletion of either the IH1(PH) or the IH2(PTB) and significantly reduced when both regions were deleted together. Thus, the PH and PTB domains equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction.     

Crystal structure of a calcium-phospholipid binding domain from cytosolic phospholipase A2

Cytosolic phospholipase A2 (cPLA2) is a calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic acid-containing membrane phospholipids to initiate the biosynthesis of eicosanoids and platelet-activating factor, potent inflammatory mediators. The calcium-dependent activation of the enzyme is mediated by an N-terminal C2 domain, which is responsible for calcium-dependent translocation of the enzyme to membranes and that enables the intact enzyme to hydrolyze membrane-resident substrates. The 2.4-A x-ray crystal structure of this C2 domain was solved by multiple isomorphous replacement and reveals a beta-sandwich with the same topology as the C2 domain from phosphoinositide-specific phospholipase C delta 1. Two clusters of exposed hydrophobic residues surround two adjacent calcium binding sites. This region, along with an adjoining strip of basic residues, appear to constitute the membrane binding motif. The structure provides a striking insight into the relative importance of hydrophobic and electrostatic components of membrane binding for cPLA2. Although hydrophobic interactions predominate for cPLA2, for other C2 domains such as in "conventional" protein kinase C and synaptotagmins, electrostatic forces prevail.     

Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain

Eps15 homology (EH) domains are eukaryotic signaling modules that recognize proteins containing Asn-Pro-Phe (NPF) sequences. The structure of the central EH domain of Eps15 has been solved by heteronuclear magnetic resonance spectroscopy. The fold consists of a pair of EF hand motifs, the second of which binds tightly to calcium. The NPF peptide is bound in a hydrophobic pocket between two alpha helices, and binding is mediated by a critical aromatic interaction as revealed by structure-based mutagenesis. The fold is predicted to be highly conserved among 30 identified EH domains and provides a structural basis for defining EH-mediated events in protein trafficking and growth factor signaling.