High-affinity interaction between the S-layer protein SbsC and the secondary cell wall polymer of Geobacillus stearothermophilus ATCC 12980 determined by surface plasmon resonance technology

Surface plasmon resonance studies using C-terminal truncation forms of the S-layer protein SbsC (recombinant SbsC consisting of amino acids 31 to 270 [rSbsC(31-270)] and rSbsC(31-443)) and the secondary cell wall polymer (SCWP) isolated from Geobacillus stearothermophilus ATCC 12980 confirmed the exclusive responsibility of the N-terminal region comprising amino acids 31 to 270 for SCWP binding. Quantitative analyses indicated binding behavior demonstrating low, medium, and high affinities.     

Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980.

During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half could function as a binding site for the hmwA.     

Structural characterization of the acid-degraded secondary cell wall polymer of Geobacillus stearothermophilus PV72/p2

The secondary cell wall polymer (SCWP) from Geobacillus stearothermophilus PV72/p2, which is involved in the anchoring of the surface-layer protein to the bacterial cell wall layer, is composed of 2-amino-2-deoxy- and 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-mannose, and 2-acetamido-2-deoxy-D-mannuronic acid. The primary structure of the acid-degraded polysaccharide--liberated by HF-treatment from the cell wall--was determined by high-field NMR spectroscopy and mass spectrometry using N-acetylated and hydrolyzed polysaccharide derivatives as well as Smith-degradation. The polysaccharide was shown to consist of a tetrasaccharide repeating unit containing a pyruvic acid acetal at a side-chain 2-acetamido-2-deoxy-alpha-D-mannopyranosyl residue. Substoichiometric substitutions of the repeating unit were observed concerning the degree of N-acetylation of glucosamine residues and the presence of side-chain linked 2-acetamido-2-deoxy-beta-D-glucopyranosyl units: [Formula: see text].     

Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS)

Nitric oxide synthase (NOS) catalyzes the NADPH- and O₂-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an N^G-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV–Vis absorption spectroscopic techniques. Spectral comparisons of ligand complexes (with O₂, NO and CO) of substrate-bound (l-Arg or NHA) gsNOS, including the key oxyferrous complex studied at ?50 °C in cryogenic mixed solvents, with analogous mammalian NOS complexes indicate overall spectroscopic similarities between gsNOS and mammalian NOSs. However, more detailed spectral comparisons reflect subtle structural differences between gsNOS and mammalian NOSs. This may be due to an incomplete tetrahydrobiopterin (BH₄)-binding site and low BH₄-binding affinity, which may become even lower in the presence of cryosolvent in gsNOS. Although BH₄-binding may be altered, gsNOS appears to require the pterin for NO production since formation of the stable ferric-NO product complex was only observed when excess BH₄ (>150 μM) over gsNOS was present upon single turnover reaction in which O₂ was bubbled into dithionite-reduced NHA-bound protein solution at ?35 °C or ?50 °C.     

The structure of an inverting GH43 beta-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues

beta-D-Xylosidases are glycoside hydrolases that catalyze the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicellulose. Here we describe the enzyme-substrate crystal structure of an inverting family 43 beta-xylosidase, from Geobacillus stearothermophilus T-6 (XynB3). Each XynB3 monomeric subunit is organized in two domains: an N-terminal five-bladed beta-propeller catalytic domain, and a beta-sandwich domain. The active site possesses a pocket topology, which is mainly constructed from the beta-propeller domain residues, and is closed on one side by a loop that originates from the beta-sandwich domain. This loop restricts the length of xylose units that can enter the active site, consistent with the exo mode of action of the enzyme. Structures of the enzyme-substrate (xylobiose) complex provide insights into the role of the three catalytic residues. The xylose moiety at the -1 subsite is held by a large number of hydrogen bonds, whereas only one hydroxyl of the xylose unit at the +1 subsite can create hydrogen bonds with the enzyme. The general base, Asp15, is located on the alpha-side of the -1 xylose sugar ring, 5.2 Angstroms from the anomeric carbon. This location enables it to activate a water molecule for a single-displacement attack on the anomeric carbon, resulting in inversion of the anomeric configuration. Glu187, the general acid, is 2.4 Angstroms from the glycosidic oxygen atom and can protonate the leaving aglycon. The third catalytic carboxylic acid, Asp128, is 4 Angstroms from the general acid; modulating its pK(a) and keeping it in the correct orientation relative to the substrate. In addition, Asp128 plays an important role in substrate binding via the 2-O of the glycon, which is important for the transition-state stabilization. Taken together, these key roles explain why Asp128 is an invariant among all five-bladed beta-propeller glycoside hydrolases.     

Coupled endo-exodeoxyribonuclease activity from HeLa cell nuclei interacts with specific regions of the SV40 mini chromosome

A recently described endo-exodeoxyribonuclease activity (1) was determined to interact preferentially with the origin/enhancer region and the 5' end of the late promoter region of the SV40 mini chromosome. The enzymatic activity has no preferential site of interaction with naked DNA, which suggests, the chromatin organization is responsible for this site directed interaction.     

Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology

The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of Geobacillus stearothermophilus PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology. The SCWP is an acidic polysaccharide that contains N-acetylglucosamine, N-acetylmannosamine, and pyruvic acid. For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layer-like homology (SLH) domain (3SLH) or the residual part of SbsB were used. Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding. The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized. Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites. The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall Kd of 7.7 x 10(-7) M. Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (Kd = 2.6 x 10(-5) M), medium (Kd = 6.1 x 10(-8) M), and high (Kd = 6.7 x 10(-11) M) affinities. Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH. Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated.     

The glycerol teichoic acid from the cell wall of Bacillus stearothermophilus B65

1. A glycerol teichoic acid has been extracted from cell walls of Bacillus stearothermophilus B65 and its structure examined. 2. Trichloroacetic acid-extractable teichoic acid accounted for 68% of the total cell-wall phosphorus and residual material could be hydrolysed to a mixture of products including those characteristic of glycerol teichoic acids. 3. The extracted polymer is composed of glycerol, phosphoric acid, d-glucose and d-alanine. 4. Hydrolysis of the polymer with alkali gave glycerol, 1-O-alpha-d-glucopyranosylglycerol and its monophosphates, glycerol mono- and di-phosphate, as well as traces of a glucosyldiglycerol triphosphate and a glucosylglycerol diphosphate. 5. The teichoic acid is a polymer of 18 or 19 glycerol phosphate units having alpha-d-glucopyranosyl residues attached to position 1 of 14 or 15 of the glycerol residues. 6. The glycerol residues are joined by phosphodiester linkages involving positions 2 and 3 in each glycerol. 7. d-Alanine is in ester linkage to the hydroxyl group at position 6 of approximately half of the glucose residues. 8. One in every 13 or 12 polymer molecules bears a phosphomonoester group on position 3 of a glucose residue, the possible significance of which in linkage of the polymer to other wall constituents is discussed.     

The structures of the cell wall teichoic acids from the thermophilic microorganism Geobacillus thermoleovorans strain Fango

The structures of two teichoic acid fractions (TA1 and TA2) isolated from the thermophilic gram-positive bacterium Geobacillus thermoleovorans strain Fango were investigated by means of chemical and NMR spectroscopic methods. The most abundant species (TA1) exhibited a rather regular structure comprising two different repeating units of 1,3-glycerol phosphate nonstoichiometrically substituted by terminal-alpha-D-Gal p (t-alpha-D-Gal p). The second molecular species (TA2) presented a higher structural variability and t-alpha-D-Glc p and the disaccharides t-alpha-D-Glc pNAc-(1-->2)-alpha-D-Glc p and t-alpha-D-Glc pNAc-(1-->3)-alpha-D-Glc p were also present as minor substituents at O-2 of the glycerol phosphate residues. Minor substitution by alanine could also be detected.     

Characterization of a new thermophilic spore photoproduct lyase from Geobacillus stearothermophilus (SplG) with defined lesion containing DNA substrates

The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S]+ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5'-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.     

The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Analysis of its glycosylation.

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n = 13-18), with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core -->3)-alpha-l-Rhap-(1-->3)-alpha-L-Rhap-(L--> and the linkage glycose beta-D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.     

Insulin-like growth factor binding protein-6 (IGFBP-6) interacts with DNA-end binding protein Ku80 to regulate cell fate

Insulin-like growth factor binding protein-6 (IGFBP-6) is a growth inhibitory protein that regulates the availability of insulin-like growth factors (IGFs). We recently reported that IGFBP-6 exerts intracellular actions via its translocation to the nucleus. We now show that IGFBP-6 co-purifies by tandem-affinity with nuclear proteins involved in DNA stability and repair such as Ku80, Ku70, histone H2B and importin-α. Furthermore, this report shows that IGFBP-6 and Ku80 interact specifically using two active binding sites for Ku80 in IGFBP-6. One of the binding sites [196RKR199], as part of the NLS-sequence in IGFBP-6 also binds importin-α which may selectively compete with Ku80 regulating its trafficking to the nucleus. Moreover, IGFBP-6 co-localized with Ku80 based on a cell cycle pattern. Overexpression of IGFBP-6 increased the nuclear Ku80 in mitotic cells and reduced it post-mitosis. It is known that if highly expressed IGFBP-6 induces apoptosis and in our model, the down-regulation of Ku80 by specific siRNAs enhanced the apoptotic effect caused by the IGFBP-6 overexpression. This study demonstrates that IGFBP-6 alters cell survival by potentially regulating the availability of Ku80 for the DNA-repair process. This action represents a novel mechanism by which growth inhibitory proteins such as IGFBP-6 regulate cell fate.     

Inhibition of the binding of penicillin to the pneumococcal penicillin-binding proteins (PBPs) by exogenous cell wall peptides

Incubation of pneumococci with D-alanine-containing peptides naturally occurring in peptidoglycan protected cells against lysis and killing by beta-lactam antibiotics near MIC. Such peptides caused decreased binding of the antibiotic to penicillin-binding proteins (PBPs), primarily PBP 2B. This provides direct evidence in vivo for the hypothesis that beta-lactams act as substrate analogues and identifies PBP 2B as a killing target in pneumococci.     

The DNA topoisomerase IIbeta binding protein 1 (TopBP1) interacts with poly (ADP-ribose) polymerase (PARP-1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.     

The Down syndrome cell adhesion molecule (DSCAM) interacts with and activates Pak

The Down syndrome cell adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily that maps to a Down syndrome region of chromosome 21q22.2-22.3. In Drosophila, Dscam functions as an axon guidance receptor regulating targeting and branching. Genetic and biochemical studies have shown that in Drosophila, Dscam activates Pak1 via the Dock adaptor molecule. The extracellular domain of human DSCAM is highly homologous to the Drosophila protein; however, the intracellular domains of both human and Drosophila DSCAM share no obvious sequence identity. To study the signaling mechanisms of human DSCAM, we investigated the interaction between DSCAM and potential downstream molecules. We found that DSCAM directly binds to Pak1 and stimulates Pak1 phosphorylation and activity, unlike Drosophila where an adaptor protein Dock mediates the interaction between Dscam and Pak1. We also observed that DSCAM activates both JNK and p38 MAP kinases. Furthermore, expression of the cytoplasmic domain of DSCAM induces a morphological change in cultured cells that is JNK-dependent. These observations suggest that human DSCAM also signals through Pak1 and may function in axon guidance similar to the Drosophila Dscam.     

The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.