Structure of the soluble domain of a membrane-anchored thioredoxin-like protein from Bradyrhizobium japonicum reveals unusual properties

TlpA is an unusual thioredoxin-like protein present in the nitrogen-fixing soil bacterium Bradyrhizobium japonicum. A hydrophobic N-terminal transmembrane domain anchors it to the cytoplasmic membrane, whereby the hydrophilic thioredoxin domain becomes exposed to the periplasmic space. There, TlpA catalyses an essential reaction, probably a reduction, in the biogenesis of cytochrome aa(3). The soluble thioredoxin domain (TlpA(sol)), devoid of the membrane anchor, was purified and crystallized. Oxidized TlpA(sol) crystallized as a non-covalent dimer in the space group P2(1)2(1)2(1). The X-ray structure analysis was carried out by isomorphous replacement using a xenon derivative. This resulted in a high-resolution (1.6 A) three-dimensional structure that displayed all of the features of a classical thioredoxin fold. A number of peculiar structural details were uncovered: (i) Only one of the two active-site-cysteine sulphurs (Cys72, the one closer to the N terminus) is exposed on the surface, making it the likely nucleophile for the reduction of target proteins. (ii) TlpA(sol) possesses a unique structural disulphide bond, formed between Cys10 and Cys155, which connects an unprecedented N-terminal alpha helix with a beta sheet near the C terminus. (iii) An insertion of about 25 amino acid residues, not found in the thioredoxin prototype of Escherichia coli, contributes only marginally to the thioredoxin fold, but forms an extra, surface-exposed alpha helix. This region plus another surface-exposed stretch (-Ile-Gly-Arg-Ala-), which is absent even in the closest TlpA relatives, might be considered as specificity determinants for the recognition of target proteins in the periplasm. The TlpA(sol) structure paves the way towards unraveling important structure-function relationships by rational mutagenesis.     

Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase

Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.     

Diffusion-ordered NMR spectroscopy: a versatile tool for the molecular weight determination of uncharged polysaccharides

Diffusion-ordered NMR spectroscopy (DOSY) experiments have been carried out on dilute aqueous solutions of uncharged saccharide systems and, in particular, on six well characterized pullulan fractions of different molecular weights. The values of diffusion coefficients and hydrodynamic radii determined for the pullulan fractions are in good agreement with the results obtained with other methodologies such as light scattering. Fitting the diffusion coefficients data as a function of the molecular weight allows for the determination of a calibration curve that can be applied to a wide range of mono-, oligo-, and polysaccharides. Therefore, DOSY is proposed as a versatile tool for achieving a simple estimation of the molecular weight of uncharged polysaccharides. Mixtures of homopolymers of different molecular weight can be nicely separated. An advantage of the method is that the same sample used for the NMR characterization can be used for the molecular weight determination without any further manipulation. Other water soluble polymers, such as poly(ethylene oxide) and poly(vinylpyrrolidone), can be roughly characterized using the same calibration curve.     

The giants of the phylum Brachiopoda: a matter of diet?

The species of the brachiopod Gigantoproductus are giants within the Palaeozoic sedentary benthos. This presents a dilemma as living brachiopods have low‐energy lifestyles. Although brachiopod metabolic rates were probably higher during the Palaeozoic than today, the massive size reached by species of Gigantoproductus is nevertheless unusual. By examining the diet of Gigantoproductus species from the Visean (Mississippian, Carboniferous) of Derbyshire (UK), we seek to understand the mechanisms that enabled those low‐metabolism brachiopod species to become giants. Were they suspension feeders, similar to all other brachiopods, or did endosymbiosis provide a lifestyle that allowed them to have higher metabolic rates and become giants? We suggest that the answer to this conundrum may be solved by the identification of the biogeochemical signatures of symbionts, through combined analyses of the carbon and nitrogen‐isotopic compositions of the occluded organic matrix within their calcite shells. The shells are formed of substructured columnar units that are remarkably long and a few hundreds of microns wide, deemed to be mostly pristine based on multiple analyses (petrography, cathodoluminescence (CL), scanning electron microscopy (SEM), electron backscatter diffraction (EBSD), transmission electron microscopy (TEM)); they contain occluded organic fractions detected by TEM, nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GC‐MS) analyses. We conclude that the gigantic size reached by the species of Gigantoproductus is probably the result of a mixotroph lifestyle, by which they could rely on the energy and nutrients derived both from photosymbiotic microbes and from filtered particulate food.     

The lipoprotein HP1454 of Helicobacter pylori regulates T‐cell response by shaping T‐cell receptor signalling

Helicobacter pylori (HP) is a Gram‐negative bacterium that chronically infects the stomach of more than 50% of human population and represents a major cause of gastric cancer, gastric lymphoma, gastric autoimmunity, and peptic ulcer. It still remains to be elucidated, which HP virulence factors are important in the development of gastric disorders. Here, we analysed the role of the HP protein HP1454 in the host–pathogen interaction. We found that a significant proportion of T cells isolated from HP patients with chronic gastritis and gastric adenocarcinoma proliferated in response to HP1454. Moreover, we demonstrated in vivo that HP1454 protein drives Th1/Th17 inflammatory responses. We further analysed the in vitro response of human T cells exposed either to an HP wild‐type strain or to a strain with a deletion of the hp1454 gene, and we revealed that HP1454 triggers the T‐cell antigen receptor‐dependent signalling and lymphocyte proliferation, as well as the CXCL12‐dependent cell adhesion and migration. Our study findings prove that HP1454 is a crucial bacterial factor that exerts its proinflammatory activity by directly modulating the T‐cell response. The relevance of these results can be appreciated by considering that compelling evidence suggest that chronic gastric inflammation, a condition that paves the way to HP‐associated diseases, is dependent on T cells.     

Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.     

Anthrax toxins inhibit immune cell chemotaxis by perturbing chemokine receptor signalling

Pathogenic strains of Bacillus anthracis produce two potent toxins, lethal toxin (LT), a metalloprotease that cleaves mitogen-activated protein kinase kinases, and oedema toxin (ET), a calcium/calmodulin-dependent adenylate cyclase. Emerging evidence indicates a role for both toxins in suppressing the initiation of both innate and adaptive immune responses, which are essential to keep the infection under control. Here we show that LT and ET inhibit chemotaxis of T-cells and macrophages by subverting signalling by both CXC and CC chemokine receptors. The data highlight a novel strategy of immunosuppression by B. anthracis based on inhibition of immune cell homing.     

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G₁ phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18–22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [³H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11–20, 1998. © 1998 Wiley-Liss, Inc.     

Crystal structure of GABA-aminotransferase, a target for antiepileptic drug therapy.

gamma-Aminobutyrate aminotransferase (GABA-AT), a pyridoxal phosphate-dependent enzyme, is responsible for the degradation of the inhibitory neurotransmitter GABA and is a target for antiepileptic drugs because its selective inhibition raises GABA concentrations in brain. The X-ray structure of pig GABA-AT has been determined to 3.0 A resolution by molecular replacement with the distantly related enzyme ornithine aminotransferase. Both omega-aminotransferases have the same fold, but exhibit side chain replacements in the closely packed binding site that explain their respective specificities. The aldimines of GABA and the antiepileptic drug vinyl-GABA have been modeled into the active site.     

Polyamine conjugate levels and ethylene biosynthesis: inverse relationship with vegetative bud formation in tobacco thin layers

The effects of two inhibitors of polyamine (spermidine and spermine) biosynthesis, cyclohexylamine (CHA; 5 and 10 mM) and methylglyoxal(bis-guanylhydrazone) (MGBG; 0.1, 0.5 and 1 mM), on the organogenic response in vegetative bud-forming tobacco (Nicotiana tabacum L. cv. Samsun) thin layer explants were evaluated micro- and macroscopically at different times during culture. The final number of buds formed and the percentage of organogenic explants was significantly reduced by both inhibitors, but much more so by MGBG than CHA. This inhibitory effect was already evident in MGBG-treated explants on day 5, in terms of the number of meristemoids per explant. On the contrary, in the presence of CHA, the number of meristemoids on day 5 was higher than that in the controls. Between days 9 and 13, meristemoid formation slowed down considerably in inhibitor-treated explants compared with controls. On day 13, the number of bud primordia was similar in control and CHA-treated explants, but significantly lower in MGBG-treated explants. This inhibitor also induced peculiar cytohistological events, such as a reduced formation of oval-shaped cell aggregates on the explant surface and more frequent cases of nucleolar extrusion, while CHA led to the appearance of hypertrophic epidermal cells; callus formation at the basal end of the explant and xylogenesis were also affected by the inhibitors. Ethylene biosynthesis, measured as [ C]methionine incorporation, was stimulated 2- (day 2) to 3-fold (15 h) by 0.5 mM MGBG, whereas CHA (10 mM) had little effect and aminoethoxyvinylglycine (AVG; 0.1 μM), an ethylene synthesis inhibitor, was strongly inhibitory. In control explants, the incorporation of labelled methionine into ethylene and spermidine followed an inverse trend up to day 8. In these explants, free putrescine increased 32-fold and spermidine increased about 10-fold between days 0 and 8. Trichloroacetic acid (TCA)-soluble conjugated putrescine also accumulated dramatically during culture. While CHA provoked a decline in spermidine levels, MGBG caused an unexpected increase in free spermidine and spermine titres; however, its most conspicuous effect was on the further enhancement of putrescine conjugate accumulation, while CHA and AVG had the opposite effect. Results are discussed in view of establishing a putative link between MGBG-enhanced ethylene synthesis, increased conjugate titres and inhibition of meristemoid formation.