Clostridium perfringensα-toxin interaction with red cells and model membranes.

The effects of Clostridium perfringensα-toxin on host cells have previously been studied extensively but the biophysical processes associated with toxicity are poorly understood. The work reported here shows that the initial interaction between the toxin and lipid membrane leads to measurable changes in the physical properties and morphology of the membrane. A Langmuir monolayer technique was used to assess the response of different lipid species to toxin. Sphingomyelin and unsaturated phosphatidylcholine showed the highest susceptibility to toxin lypolitic action, with a two stage response to the toxin (an initial, rapid hydrolysis stage followed by the insertion and/or reorganisation of material in the monolayer). Fluorescence confocal microscopy on unsaturated phosphatidylcholine vesicles shows that the toxin initially aggregates at discrete sites followed by the formation of localised "droplets" accumulating the hydrolysis products. This process is accompanied by local increases in the membrane dipole potential by about 50 (±42) mV. In contrast, red blood cells incubated with the toxin suffered a decrease of the membrane dipole potential by 50 (±40) mV in areas of high toxin activity (equivalent to a change in electric field strength of 10(7) V m(-1)) which is sufficient to affect the functioning of the cell membrane. Changes in erythrocyte morphology caused by the toxin are presented, and the early stages of interaction between toxin and membrane are characterised using thermal shape fluctuation analysis of red cells which revealed two distinct regimes of membrane-toxin interaction.

Crystal structure of R120G disease mutant of human αB-crystallin domain dimer shows closure of a groove.

Small heat shock proteins form large cytosolic assemblies from an "α-crystallin domain" (ACD) flanked by sequence extensions. Mutation of a conserved arginine in the ACD of several human small heat shock protein family members causes many common inherited diseases of the lens and neuromuscular system. The mutation R120G in αB-crystallin causes myopathy, cardiomyopathy and cataract. We have solved the X-ray structure of the excised ACD dimer of human αB R120G close to physiological pH and compared it with several recently determined wild-type vertebrate ACD dimer structures. Wild-type excised ACD dimers have a deep groove at the interface floored by a flat extended "bottom sheet." Solid-state NMR studies of large assemblies of full-length αB-crystallin have shown that the groove is blocked in the ACD dimer by curvature of the bottom sheet. The crystal structure of R120G ACD dimer also reveals a closed groove, but here the bottom sheet is flat. Loss of Arg120 results in rearrangement of an extensive array of charged interactions across this interface. His83 and Asp80 on movable arches on either side of the interface close the groove by forming two new salt bridges. The residues involved in this extended set of ionic interactions are conserved in Hsp27, Hsp20, αA- and αB-crystallin sequences. They are not conserved in Hsp22, where mutation of the equivalent of Arg120 causes neuropathy. We speculate that the αB R120G mutation disturbs oligomer dynamics, causing the growth of large soluble oligomers that are toxic to cells by blocking essential processes.

Comparison of a nontoxic variant of Clostridium perfringens α-toxin with the toxic wild-type strain.

The α-toxin produced by Clostridium perfringens is one of the best-studied examples of a toxic phospholipase C. In this study, a nontoxic mutant protein from C. perfringens strain NCTC8237 in which Thr74 is substituted by isoleucine (T74I) has been characterized and is compared with the toxic wild-type protein. Thr74 is part of an exposed loop at the proposed membrane-interfacing surface of the toxin. The mutant protein had markedly reduced cytotoxic and myotoxic activities. However, this substitution did not significantly affect the catalytic activity towards water-soluble substrate or the overall three-dimensional structure of the protein. The data support the proposed role of the 70-90 loop in the recognition of membrane phospholipids. These findings also provide key evidence in support of the hypothesis that the hydrolysis of both phosphatidylcholine and sphingomyelin are required for the cytolytic and toxic activity of phospholipases.

Crystal structures of alpha-crystallin domain dimers of alphaB-crystallin and Hsp20.

Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised alpha-crystallin domain from rat Hsp20 and that from human alphaB-crystallin show that they form homodimers with a shared groove at the interface by extending a beta sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the alphaB-crystallin interface show that it has a high density of positive charges. The disease mutant R120G alpha-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.

Modified constraint-induced movement therapy for young children with hemiplegic cerebral palsy: a pilot study.

The objective of the study was to evaluate the effectiveness of modified constraint-induced movement therapy in young children with hemiplegia. It was a single-case experimental design using children as their own controls. Assessment was at entry to the study and subsequently at 4-weekly intervals. A 4-week baseline period with no hand treatment, controlling for maturation, was followed by a 4-week treatment period and a second 4-week period with no hand treatment to measure carry-over. Treatment consisted of twice-weekly 1-hour sessions of structured activities with a therapist and a home programme for non-treatment days. Only verbal instruction and gentle restraint of the unaffected arm were used to encourage use of the affected arm. Nine children (six males, three females; median age 31 mo, age range 21 to 61 mo) presenting with congenital spastic hemiplegia (five right side, four left side) were involved in the study. Changes in hand function were evaluated with the Quality of Upper Extremity Skills Test. Improvement was seen throughout the study with statistical significance, using the Wilcoxon signed rank test, of 0.01 immediately after treatment. Results of this pilot study suggest that this modification of constraint-induced movement therapy may be an effective way of treating young children with hemiplegia. Future work is planned to consolidate and develop these results.

NADP+ and NAD+ binding to the dual coenzyme specific enzyme Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: different interdomain hinge angles are seen in different binary and ternary complexes.

The reduced coenzymes NADH and NADPH only differ by one phosphate, but in the cell NADH provides reducing power for catabolism while NADPH is utilized in biosynthetic pathways. Enzymes almost invariably discriminate between the coenzymes, but glucose 6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides is rare in being functionally dual specific. In order to elucidate the coenzyme selectivity, the structures of NADP(+)- and NAD(+)-complexed L. mesenteroides G6PD have been determined including data to 2.2 and 2.5 A resolution, respectively, and compared with unliganded G6PD crystallized in the same space groups. Coenzyme binding is also compared with that in a ternary complex of a mutant in which Asp177 in the active site has been mutated to asparagine. There are no gross structural differences between the complexes. In both binary complexes, the enzyme interdomain hinge angle has opened. NADP(+) binds to the furthest open form; of the residues within the coenzyme domain, only Arg46 moves, interacting with the 2'-phosphate and adenine. NAD(+) is less well defined in the binding site; smaller hinge opening is seen but larger local changes: Arg46 is displaced, Thr14 bonds the 3'-hydroxyl and Gln47 bonds the 2'-hydroxyl. In the ternary complex, the hinge angle has closed; only the adenine nucleotide is ordered in the binding site. Arg46 again provides most binding interactions.

Opening of the active site of Clostridium perfringens alpha-toxin may be triggered by membrane binding.

On the basis of amino acid sequence homologies with other phospholipases C, the alpha-toxin of Clostridium perfringens was predicted to be a two-domain protein. Using truncated forms of alpha-toxin the phospholipase C active site was shown to be located in the amino-terminal domain. Crystallographic studies have confirmed this organisation and have also revealed that the carboxy-terminal domain is structurally similar to the phospholipid-binding domains in eukaryotic proteins. This information has been used to devise a model predicting how alpha-toxin interacts with membranes via calcium-mediated recognition of phospholipid head groups and the interaction of hydrophobic amino acids with the phospholipid tail group. The binding of alpha-toxin to membranes appears to result in the opening of the active site allowing hydrolysis of membrane phospholipids.

An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme.

The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.

Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities.

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.

Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin.

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholipase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium ions have been shown to be required for the specific binding of toxin to membranes prior to phospholipid cleavage. Reported X-ray crystallographic structures of the toxin show that the C-terminal domain has a fold that is analogous to the eukaryotic calcium and membrane-binding C2 domains. We report the binding sites for three calcium ions that have been identified, by crystallographic methods, in the C-terminal domain of the protein close to the postulated membrane-binding surface. The position of these ions at the tip of the domain, and their function (to facilitate membrane binding) is similar to that of calcium ions observed bound to C2 domains. Using the optical spectroscopic techniques of circular dichroism (CD) and fluorescence spectroscopy, pronounced changes to both near and far-UV CD and tryptophan emission fluorescence upon addition of calcium to the C-terminal domain of alpha-toxin have been observed. The changes in near-UV CD, fluorescence enhancement and a 2 nm blue-shift in the fluorescence emission spectrum are consistent with tryptophan residue(s) becoming more immobilised in a hydrophobic environment. Calcium binding appears to be low-affinity: Kd approximately 175-250 microM at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic methods.

Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement.

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 A resolution data set and a dimer of the large beta + alpha domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 A. Crystals of the deletion mutant DeltaG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 A resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.

The Clostridium perfringens alpha-toxin.

The gene encoding the alpha-(cpa) is present in all strains of Clostridium perfringens, and the purified alpha-toxin has been shown to be a zinc-containing phospholipase C enzyme, which is preferentially active towards phosphatidylcholine and sphingomyelin. The alpha-toxin is haemolytic as a result if its ability to hydrolyse cell membrane phospholipids and this activity distinguishes it from many other related zinc-metallophospholipases C. Recent studies have shown that the alpha-toxin is the major virulence determinant in cases of gas gangrene, and the toxin might play a role in several other diseases of animals and man as diverse as necrotic enteritis in chickens and Crohn's disease in man. In gas gangrene the toxin appears to have three major roles in the pathogenesis of disease. First, it is able to cause mistrafficking of neutrophils, such that they do not enter infected tissues. Second, the toxin is able to cause vasoconstriction and platelet aggregation which might reduce the blood supply to infected tissues. Finally, the toxin is able to detrimentally modulate host cell metabolism by activating the arachidonic acid cascade and protein kinase C. The molecular structure of the alpha-toxin reveals a two domain protein. The amino-terminal domain contains the phospholipase C active site which contains zinc ions. The carboxyterminal domain is a paralogue of lipid binding domains found in eukaryotes and appears to bind phospholipids in a calcium-dependent manner. Immunisation with the non-toxic carboxyterminal domain induces protection against the alpha-toxin and gas gangrene and this polypeptide might be exploited as a vaccine. Other workers have exploited the entire toxin as the basis of an anti-tumour system.

Structure of the key toxin in gas gangrene.

Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

The C-terminal domains of gammaS-crystallin pair about a distorted twofold axis.

The 2-domain gammaS-crystallin, a highly conserved early evolutionary off-shoot of the gamma-crystallin family, is located in the water-rich region of eye lenses. The expressed C-terminal domain, gammaS-C, has been crystallized and the 2.56 A X-ray structure determined. There are two domains in the asymmetric unit which pair about a distorted twofold axis. One of the domains has an altered conformation in a highly conserved region of the protein, the tyrosine corner. The distorted gammaS-C dimer of domains is compared with the highly symmetrical, equivalent recombinant dimer of C-terminal domains from gammaB-crystallin. Sequence changes close to the interface, that distinguish gammaS from the other gamma-crystallins, are examined in order to evaluate their role in symmetrical domain pairing.

Amino acid substitutions at the dimer interface of human glucose-6-phosphate dehydrogenase that increase thermostability and reduce the stabilising effect of NADP.

Over 100 mutations of the G6PD gene have been documented. With the construction of the molecular model of glucose-6-phosphate dehydrogenase, based on the structure of the bacterial Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, it has been possible to superimpose these amino acid changes on to the structure of the glucose-6-phosphate dehydrogenase molecule. There are a large number of severe disease causing mutations at the dimer interface which usually cause decreased thermostability. We have used this knowledge to predict amino acid changes which would effect an increase in the stability of the dimer. The aspartic acid at residue 421 was chosen as it is a negatively charged residue at the centre of the dimer interface in an area rich in negatively charged residues. This residue was changed to a neutrally charged alanine or asparagine, or a positively charged lysine or arginine. The thermostability of the enzyme was increased when residue 421 was neutral (A or N) and increased further when positive (K or R). NADP is known to exert a concentration dependent stabilising effect on the glucose-6-phosphate dehydrogenase dimer. However the concentration-dependent stabilising effect of NADP was reduced in the residue-421 substitutions in a manner which was inversely proportional to charge change. These results suggest that changes at the dimer interface can also affect the distant (> 20 A) NADP-binding site, and vice versa; an attempt has been made to explain these interactions based on the molecular model of human glucose-6-phosphate dehydrogenase.

Crystallization and preliminary X-ray diffraction studies of alpha-toxin from two different strains (NCTC8237 and CER89L43) of Clostridium perfringens.

The alpha-toxin of Clostridium perfringens is the major virulence determinant for gas gangrene in man. The gene encoding the alpha-toxin has been cloned into E. coli from two strains of the bacterium (NCTC8237 and CER89L43) and subsequently purified to homogeneity. The two strains of alpha-toxin differ by five amino acids, resulting in the toxin from NCTC8237 being sensitive to chymotrypsin digestion while that from CER89L43 is resistant. The alpha-toxin from each of these strains has been crystallized in two different forms by the hanging-drop vapour-diffusion method at 293 K. CER89L43 form I crystals belong to space group R32 and have two molecules in the crystallographic asymmetric unit and a unit cell with a = b = 151.4, c = 195.5 A, alpha = beta = 90, gamma = 120 degrees. The crystals diffracted to dmin = 1.90 A. The characteristics of the NCTC8237 form I crystals have already been reported. The form II crystals from both strains belong to space group C2221 with one molecule in the crystallographic asymmetric unit and, for strain CER89L43, have cell dimensions a = 61.05, b = 177.50, c = 79.05 A, alpha = beta = gamma = 90 degrees, while for strain NCTC8237 the cell dimensions are a = 60.50, b = 175.70, c = 80.20 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to maximum resolutions of 1.85 and 2.1 A for the CER89L43 and the NCTC8237 strains, respectively.

Glucose 6-phosphate dehydrogenase mutations causing enzyme deficiency in a model of the tertiary structure of the human enzyme.

Human glucose 6-phosphate dehydrogenase (G6PD) has a particularly large number of variants resulting from point mutations; some 60 mutations have been sequenced to date. Many variants, some polymorphic, are associated with enzyme deficiency. Certain variants have severe clinical manifestations; for such variants, the mutant enzyme almost always displays a reduced thermal stability. A homology model of human G6PD has been built, based on the three-dimensional structure of the enzyme from Leuconostoc mesenteroides. The model has suggested structural reasons for the diminished enzyme stability and hence for deficiency. It has shown that a cluster of mutations in exon 10, resulting in severe clinical symptoms, occurs at or near the dimer interface of the enzyme, that the eight-residue deletion in the variant Nara is at a surface loop, and that the two mutations in the A- variant are close together in the three-dimensional structure.

Event related potentials in adults diagnosed as reading disabled in childhood.

The purpose of this study was to identify electrophysiological correlates of reading disability (RD) in adults with psychometrically documented childhood reading histories. Specific a-priori hypotheses for these correlates were generated from the findings of Harter, Anllo-Vento, Wood & Schroeder (1988a); Harter, Diering & Wood (1988b). The subjects were 32 males with normal intelligence and no history of attention deficit disorder or current major psychopathology. Event related potentials were recorded over O1, O2, C3', C4', F3, and F4 to letter stimuli using an intralocation selective attention paradigm. Subjects with RD showed a general reduction in positivity starting at 150 ms and continuing up to 500 ms. The reduced positivity at left central P240 replicated the findings of Harter et al. (1988b) with children. However, adult RD was associated with more diffuse, bilateral reduction in electrophysiological response to all stimuli. A possible relationship between the bilaterality of the neural deficit and the severity of the dyslexia was proposed: that a bilateral deficit may be involved in severe cases.