Role of metals in the biological activity of Clostridium botulinum neurotoxins

Clostridium botulinum neurotoxins are the most potent toxins to humans and cause paralysis by blocking neurotransmitter release at the presynaptic nerve terminals. The toxicity involves four steps, viz., binding to neuronal cells, internalization, translocation, and catalytic activity. While the catalytic activity is a zinc endopeptidase activity on the SNARE complex proteins, the translocation is believed to be a pH-dependent process allowing the translocation domain to change its conformation to penetrate the endosomal membrane. Here, we report the crystal structures of botulinum neurotoxin type B at various pHs and of an apo form of the neurotoxin, and discuss the role of metal ions and the effect of pH variation in the biological activity. Except for the perturbation of a few side chains, the conformation of the catalytic domain is unchanged in the zinc-depleted apotoxin, suggesting that zinc's role is catalytic. We have also identified two calcium ions in the molecule and present biochemical evidence to show that they play a role in the translocation of the light chain through the membrane.     

Occurrence of Vibrio parahaemolyticus and Its Specific Phages from Shrimp Ponds in East Coast of India

Vibrio parahaemolyticus is a natural microflora of marine and coastal water bodies and associated with mortality of larval shrimp in penaeid shrimp in ponds. Bacteriophages occur virtually in all places where their hosts exist. In this study, total distribution of V. parahaemolyticus and its phages were examined in shrimp ponds, seawater, estuary, animal surface, and tissues. Total vibrio count in sediments of two ponds was found to be 2.6 × 10³ and 5.6 × 10³ cfu/g. Incidence of V. parahaemolyticus in the ponds was close, while it was markedly higher in the animal surface and tissue samples. Biochemically identified eight strains of V. parahaemolyticus (V1, V3–V6, V9, V11, and V12) were taken for further infection studies with bacteriophage. Totally five bacteriophages capable of infecting V. parahaemolyticus MTCC-451 strain were isolated from all the samples. One of the isolated bacteriophage Vp1 from estuary was able to lyse all the isolated V. parahaemolyticus strains we used. Therefore, the morphology of Vp1 was estimated in TEM. Vp1 phage head measuring approximately about 50–60 nm diameter with icosahedral outline and a contractile tails of diameter 7 nm and length 100 nm and it was identified as Myoviridae. Therefore, the phages have the potential application in destroying bacterial pathogens.     

BIND – An algorithm for loss-less compression of nucleotide sequence data

Recent advances in DNA sequencing technologies have enabled the current generation of life science researchers to probe deeper into the genomic blueprint. The amount of data generated by these technologies has been increasing exponentially since the last decade. Storage, archival and dissemination of such huge data sets require efficient solutions, both from the hardware as well as software perspective. The present paper describes BIND-an algorithm specialized for compressing nucleotide sequence data. By adopting a unique 'block-length' encoding for representing binary data (as a key step), BIND achieves significant compression gains as compared to the widely used general purpose compression algorithms (gzip, bzip2 and lzma). Moreover, in contrast to implementations of existing specialized genomic compression approaches, the implementation of BIND is enabled to handle non-ATGC and lowercase characters. This makes BIND a loss-less compression approach that is suitable for practical use. More importantly, validation results of BIND (with real-world data sets) indicate reasonable speeds of compression and decompression that can be achieved with minimal processor/ memory usage. BIND is available for download at http://metagenomics.atc.tcs.com/compression/BIND. No license is required for academic or non-profit use.     

The biofilm formation defect of a Bacillus subtilis flotillin‐defective mutant involves the protease FtsH

Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix‐producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft‐specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal‐localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.     

Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

Economically viable production of solvents through acetone-butanol-ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of five proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.     

Heterologous expression and purification of Arabidopsis thaliana VIM1 protein: in vitro evidence for its inability to recognize hydroxymethylcytosine, a rare base in Arabidopsis DNA

The discovery of 5-hydroxymethyl-cytosine (5hmC) in mammalian cells prompted us to look for this base in the DNA of Arabidopsis thaliana (thale cress), and to ask how well the Arabidopsis Variant in Methylation 1 (VIM1) protein, an essential factor in maintaining 5-cytosine methylation (5mC) homeostasis and epigenetic silencing in this plant, recognizes this novel base. We found that the DNA of Arabidopsis' leaves and flowers contain low levels of 5hmC. We also cloned and expressed in Escherichia coli full-length VIM1 protein, the archetypal member of the five Arabidopsis VIM gene family. Using in vitro binding assays, we observed that full-length VIM1 binds preferentially to hemi-methylated DNA with a single modified 5mCpG site; this result is consistent with its known role in preserving DNA methylation in vivo following DNA replication. However, when 5hmC replaces one or both cytosine residues at a palindromic CpG site, VIM1 binds with approximately ≥10-fold lower affinity. These results suggest that 5hmC may contribute to VIM-mediated passive loss of cytosine methylation in vivo during Arabidopsis DNA replication.     

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.     

Crystal structure of Mycobacterium tuberculosis YefM antitoxin reveals that it is not an intrinsically unstructured protein

Toxin-antitoxin modules are present on chromosomes of almost all free-living prokaryotes. Some are implicated to act as stress-responsive elements, among their many functional roles. The YefM-YoeB toxin-antitoxin system is present in many bacterial species, where YefM belongs to the Phd family antidote of phage P1, whereas YoeB is a homolog of the RelE toxin of the RelBE system, rather than the Doc system of phage P1. YoeB, a ribonuclease, is believed to be conformationally stable, whereas YefM has been proposed to be a member of intrinsically disordered proteins. The ribonucleolytic activity of YoeB is neutralized by YefM upon formation of the YefM-YoeB complex. We report here the crystal structure of Mycobacterium tuberculosis YefM from two crystal isoforms. Our crystallographic and biophysical studies reveal that YefM is not an intrinsically unfolded protein and instead forms a well-defined structure with significant secondary and tertiary structure conformations. The residues involved in core formation of the folded structure are evolutionarily conserved among many bacterial species, supporting our observation. The C-terminal end of its polypeptide is highly pliable, which adopts different conformations in different monomers. Since at the physiological level YefM controls the activity of YoeB through intricate protein-protein interactions, the conformational heterogeneity in YefM revealed by our structure suggests that these might act a master switch in controlling YoeB activity.     

Chromatin dynamics and gene positioning

The mammalian cell nucleus provides a landscape where genes are regulated through their organization and association with freely diffusing proteins and nuclear domains. In many cases, specific genes are highly dynamic, and the principles governing their movements and interchromosomal interactions are currently under intensive study. Recent investigations have implicated actin and myosin in chromatin dynamics and gene expression. Here, we discuss our current understanding of the dynamics of the interphase genome and how it impacts nuclear organization and gene activity.     

Heparin-induced cis- and trans-dimerization modes of the thrombospondin-1 N-terminal domain

Through its interactions with proteins and proteoglycans, thrombospondin-1 (TSP-1) functions at the interface of the cell membrane and the extracellular matrix to regulate matrix structure and cellular phenotype. We have previously determined the structure of the high affinity heparin-binding domain of TSP-1, designated TSPN-1, in association with the synthetic heparin, Arixtra. To establish that the binding of TSPN-1 to Arixtra is representative of the association with naturally occurring heparins, we have determined the structures of TSPN-1 in complex with heparin oligosaccharides containing eight (dp8) and ten (dp10) subunits, by x-ray crystallography. We have found that dp8 and dp10 bind to TSPN-1 in a manner similar to Arixtra and that dp8 and dp10 induce the formation of trans and cis TSPN-1 dimers, respectively. In silico docking calculations partnered with our crystal structures support the importance of arginine residues in positions 29, 42, and 77 in binding sulfate groups of the dp8 and dp10 forms of heparin. The ability of several TSPN-1 domains to bind to glycosaminoglycans simultaneously probably increases the affinity of binding through multivalent interactions. The formation of cis and trans dimers of the TSPN-1 domain with relatively short segments of heparin further enhances the ability of TSP-1 to participate in high affinity binding to glycosaminoglycans. Dimer formation may also involve TSPN-1 domains from two separate TSP-1 molecules. This association would enable glycosaminoglycans to cluster TSP-1.