Khaja Faisal Tarique

Concentration-dependent antagonistic persuasion of SDS and naphthalene derivatives on the fibrillation of stem bromelain

Sodium dodecyl sulfate, a biological membrane mimetic, can be used to study the conversion of globular proteins into amyloid fibrils in vitro. Using multiple approaches, the effect of SDS was examined on stem bromelain (SB), a widely recognized therapeutic protein. SB is known to exist as a partially folded intermediate at pH 2.0, situation also encountered in the gastrointestinal tract (its site of absorption). In the presence of sub-micellar SDS concentration (500-1000 μM), this intermediate was found to exhibit great propensity to form large-sized β-sheeted aggregates with fibrillar morphology, the hall marks of amyloid structure. We also observed inhibition of fibrillation by two naphthalene-based compounds, ANS and bis-ANS. While bis-ANS significantly inhibited fibril formation at 50 μM, ANS did so at relatively higher concentration (400 μM). Alcohols, but not salts, were found to weaken the inhibitory action of these compounds suggesting the possible involvement of hydrophobic interactions in their binding to protein. Besides, isothermal titration calorimetry and molecular docking studies suggested that inhibition of fibrillation by these naphthalene derivatives is mediated not just through hydrophobic forces, but also by disruption of π-π interactions between the aromatic residues together with the inter-polypeptide chain repulsion among negatively charged ANS/bis-ANS bound SB.

Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori

Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp.

Crystal structure of HINT from Helicobacter pylori

Proteins belonging to the histidine triad (HIT) superfamily bind nucleotides and use the histidine triad motif to carry out dinucleotidyl hydrolase, nucleotidyltransferase and phosphoramidite hydrolase activities. Five different branches of this superfamily are known to exist. Defects in these proteins in humans are linked to many diseases such as ataxia, diseases of RNA metabolism and cell-cycle regulation, and various types of cancer. The histidine triad nucleotide protein (HINT) is nearly identical to proteins that have been classified as protein kinase C-interacting proteins (PKCIs), which also have the ability to bind and inhibit protein kinase C. The structure of HINT, which exists as a homodimer, is highly conserved from humans to bacteria and shares homology with the product of fragile histidine triad protein (FHit), a tumour suppressor gene of this superfamily. Here, the structure of HINT from Helicobacter pylori (HpHINT) in complex with AMP is reported at a resolution of 3 Å. The final model has R and Rfree values of 26 and 28%, respectively, with good electron density. Structural comparison with previously reported homologues and phylogenetic analysis shows H. pylori HINT to be the smallest among them, and suggests that it branched out separately during the course of evolution. Overall, this structure has contributed to a better understanding of this protein across the animal kingdom.

Structural characterization and functional analysis of Cystathionine β‐synthase: an enzyme involved in the reverse transsulfuration pathway of Bacillus anthracis

The reverse transsulfuration pathway has been reported to produce cysteine from homocysteine in eukaryotes ranging from protozoans to mammals while bacteria and plants produce cysteine via a de novo pathway. Interestingly, the bacterium Bacillus anthracis includes enzymes of the reverse transsulfuration pathway viz. cystathionine β‐synthase [BaCBS, previously annotated to be an O‐acetylserine sulfhydrylase (OASS)] and cystathionine γ‐lyase. Here, we report the structure of BaCBS at a resolution of 2.2 Å. The enzyme was found to show CBS activity only with activated serine (O‐acetylserine) and not with serine, and was also observed to display OASS activity but not serine sulfhydrylase activity. BaCBS was also found to produce hydrogen sulfide (H2S) upon reaction of cysteine and homocysteine. A mutational study revealed Glu 220, conserved in CBS, to be necessary for generating H2S. Structurally, BaCBS display a considerably more open active site than has been found for any other CBS or OASS, which was attributed to the presence of a helix at the junction of the C‐ and N‐terminal domains. The root‐mean‐square deviation (RMSD) between the backbone Cα carbon atoms of BaCBS and those of other CBSs and OASSs were calculated to be greater than 3.0 Å. The pyridoxal 5′‐phosphate at the active site was not traced, and appeared to be highly flexible due to the active site being wide open. Phylogenetic analysis revealed the presence of an O‐acetylserine‐dependent CBS in the bacterial domain and making separate clade from CBS and OASS indicating its evolution for specific function.

Structural and functional insights into the stationary-phase survival protein SurE, an important virulence factor of Brucella abortus

The stationary-phase survival protein SurE from Brucella abortus (BaSurE) is a metal-dependent phosphatase that is essential for the survival of this bacterium in the stationary phase of its life cycle. Here, BaSurE has been biochemically characterized and its crystal structure has been determined to a resolution of 1.9 Å. BaSurE was found to be a robust enzyme, showing activity over wide ranges of temperature and pH and with various phosphoester substrates. The active biomolecule is a tetramer and each monomer was found to consist of two domains: an N-terminal domain, which forms an approximate α + β fold, and a C-terminal domain that belongs to the α/β class. The active site lies at the junction of these two domains and was identified by the presence of conserved negatively charged residues and a bound Mg(2+) ion. Comparisons of BaSurE with its homologues have revealed both common features and differences in this class of enzymes. The number and arrangement of some of the equivalent secondary structures, which are seen to differ between BaSurE and its homologues, are responsible for a difference in the size of the active-site area and the overall oligomeric state of this enzyme in other organisms. As it is absent in mammals, it has the potential to be a drug target.

Response to Errors in Crystal structure of HINT from Helicobacter pylori

A response to the article by Maize [(2016), Acta Cryst. F72, 336-337].