Sonika Bhatnagar

PemK Toxin of Bacillus anthracis Is a Ribonuclease AN INSIGHT INTO ITS ACTIVE SITE, STRUCTURE, AND FUNCTION

Bacillus anthracis genome harbors a toxin-antitoxin (TA) module encoding pemI (antitoxin) and pemK (toxin). This study describes the rPemK as a potent ribonuclease with a preference for pyrimidines (C/U), which is consistent with our previous study that demonstrated it as a translational attenuator. The in silico structural modeling of the PemK in conjunction with the site-directed mutagenesis confirmed the role of His-59 and Glu-78 as an acid-base couple in mediating the ribonuclease activity. The rPemK is shown to form a complex with the rPemI, which is in line with its function as a TA module. This rPemI-rPemK complex becomes catalytically inactive when both the proteins interact in a molar stoichiometry of 1. The rPemI displays vulnerability to proteolysis but attains conformational stability only upon rPemK interaction. The pemI-pemK transcript is shown to be up-regulated upon stress induction with a concomitant increase in the amount of PemK and a decline in the PemI levels, establishing the role of these modules in stress. The artificial perturbation of TA interaction could unleash the toxin, executing bacterial cell death. Toward this end, synthetic peptides are designed to disrupt the TA interaction. The peptides are shown to be effective in abrogating TA interaction in micromolar range in vitro. This approach can be harnessed as a potential antibacterial strategy against anthrax in the future.

Functional characterization of WalRK: A two-component signal transduction system from Bacillus anthracis☆

Two‐component signal transduction systems (TCS), consisting of a sensor histidine protein kinase and its cognate response regulator, are an important mode of environmental sensing in bacteria. Additionally, they have been found to regulate virulence determinants in several pathogens. Bacillus anthracis, the causative agent of anthrax and a bioterrorism agent, harbours 41 pairs of TCS. However, their role in its pathogenicity has remained largely unexplored. Here, we show that WalRK of B. anthracis forms a functional TCS which exhibits some species‐specific functions. Biochemical studies showed that domain variants of WalK, the histidine kinase, exhibit classical properties of autophosphorylation and phosphotransfer to its cognate response regulator WalR. Interestingly, these domain variants also show phosphatase activity towards phosphorylated WalR, thereby making WalK a bifunctional histidine kinase/phosphatase. An in silico regulon determination approach, using a consensus binding sequence from Bacillus subtilis, provided a list of 30 genes that could form a putative WalR regulon in B. anthracis. Further, electrophoretic mobility shift assay was used to show direct binding of purified WalR to the upstream regions of three putative regulon candidates, an S‐layer protein EA1, a cell division ABC transporter FtsE and a sporulation histidine kinase KinB3. Our work lends insight into the species‐specific functions and mode of action of B. anthracis WalRK.

The role of dehydro-Alanine in the design of peptides

X-ray crystallography, NMR spectroscopy and theoretical studies on some oligopeptides containing dehydro-alanine (delta Ala) have indicated that delta Ala adopts an extended conformation and also induces a definite conformation in the preceding saturated residue. In order to evaluate the conformational constraints imposed by delta Ala on the neighbouring saturated residues, we have undertaken a systematic, theoretical study of the preferred conformations of tripeptide sequences of the type N-Ac-X-delta Ala-NHCH3 and N-Ac-delta Ala-X-NHCH3 (X = Gly, L-Ala, L-Val, L-Ile and L-Phe). The methodology and parameters used have been standardized against sequences with known crystal structures. The significant findings of this study are that delta Ala always adopts an extended conformation and induces in both the preceding and the succeeding neighbouring saturated residues a conformation in which phi approximately 140 degrees and psi approximately -40 degrees. These results have a direct application in the design of peptide sequences for specific biological activity.

Linkage, Mobility, and Selfishness in the MazF Family of Bacterial Toxins: A Snapshot of Bacterial Evolution

Prokaryotic MazF family toxins cooccur with cognate antitoxins having divergent DNA-binding folds and can be of chromosomal or plasmid origin. Sequence similarity search was carried out to identify the Toxin–Antitoxin (TA) operons of MazF family followed by sequence analysis and phylogenetic studies. The genomic DNA upstream of the TA operons was searched for the presence of regulatory motifs. The MazF family toxins showed a conserved hydrophobic pocket in a multibinding site and are present in pathogenic bacteria. The toxins of the MazF family are associated with four main types of cognate antitoxin partners and cluster as a subfamily on the branches of the phylogenetic tree. This indicates that transmission of the entire operon is the dominant mode of inheritance. The plasmid borne TA modules were interspersed between the chromosomal TA modules of the same subfamily, compatible with a frequent interchange of TA genes between the chromosome and the plasmid akin to that observed for antibiotic resistance gens. The split network of the MazF family toxins showed the AbrB-linked toxins as a hub of horizontal gene transfer. Distinct motifs are present in the upstream region of each subfamily. The presence of MazF family TA modules in pathogenic bacteria and identification of a conserved binding pocket are significant for the development of novel antibacterials to disrupt the TA interaction. However, the role of TAs in stress resistance needs to be established. Phylogenetic studies provide insight into the evolution of MazF family TAs and effect on the bacterial genome.

Structure-based design of a novel peptide inhibitor of HIV-1 integrase: a computer modeling approach.

Abstract The insertion of viral DNA into the host chromosome is an essential step in the replication of HIV-1, and is carried out by an enzyme, HIV-1 integrase (IN). Since the latter has no human cellular counterpart, it is an attractive target for antiviral drug design. Several IN inhibitors having activities in the micromolar range have been reported to date. However, no clinically useful inhibitors have yet been developed. Recently reported diketo acids represent a novel and selective class of IN inhibitors. These are the only class which appear to selectively target integrase and two of the inhibitors, L-708,906 and L-731,988, are the most potent inhibitors of preintegration complexes described to date.

Integrative analysis of ocular complications in atherosclerosis unveils pathway convergence and crosstalk.

Abstract Atherosclerosis is a life-threatening disease and a major cause of mortalities worldwide. While many of the atherosclerotic sequelae are reflected as microvascular effects in the eye, the molecular mechanisms of their development is not yet known. In this study, we employed a systems biology approach to unveil the most significant events and key molecular mediators of ophthalmic sequelae caused by atherosclerosis. Literature mining was used to identify the proteins involved in both atherosclerosis and ophthalmic diseases. A protein–protein interaction (PPI) network was prepared using the literature-mined seed nodes. Network topological analysis was carried out using Cytoscape, while network nodes were annotated using database for annotation, visualization and integrated discovery in order to identify the most enriched pathways and processes. Network analysis revealed that mitogen-activated protein kinase 1 (MAPK1) and protein kinase C occur with highest betweenness centrality, degree and closeness centrality, thus reflecting their functional importance to the network. Our analysis shows that atherosclerosis-associated ophthalmic complications are caused by the convergence of neurotrophin signaling pathways, multiple immune response pathways and focal adhesion pathway on the MAPK signaling pathway. The PPI network shares features with vasoregression, a process underlying multiple vascular eye diseases. Our study presents a first clear and composite picture of the components and crosstalk of the main pathways of atherosclerosis-induced ocular diseases. The hub bottleneck nodes highlight the presence of molecules important for mediating the ophthalmic complications of atherosclerosis and contain five established drug targets for future therapeutic modulation efforts.

Mechanisms Underlying the Opposing Effects of P2Y Receptors on the Cell Cycle

The purinergic P2Y receptors are critical determinants of physiological function playing diverse roles in cell proliferation, differentiation, and survival. However, in cancer cells they have been reported to assume contradictory roles at times, thus causing inhibition of proliferation, cell death, and apoptosis. A critical evaluation and analysis of the existing experimental data show that this response is caused by multiple mechanisms at the level of the ligand, receptor, G protein, intracellular signaling, such as the mitogen activated protein kinase pathways and surface expression. This review addresses the impact of these factors in determining the outcome of P2Y receptor activation. Working in conjunction, these factors can act to produce the observed opposing effects on the cell cycle. Thus, they are important factors in the pathogenesis and control of cancer.

In silico structure-based design of a potent, mutation resilient, small peptide inhibitor of HIV-1 reverse transcriptase.

Abstract A crucial step in the replication of HIV-1 is the conversion of its single-stranded RNA to double-stranded DNA, which is catalyzed by the virally encoded reverse transcriptase (RT). The latter is therefore a key target for the development of anti-HIV drugs. Currently approved anti- RT drugs fall into two main classes: (i) nucleoside analog inhibitors which are incorporated into the primer strand in their metabolically activated triphosphate forms, causing termination of DNA synthesis due to their 3′-deoxy configuration and (ii) the non-nucleoside inhibitors (NNIs), which are generally specific for HIV-1 RT and bind at an allosteric site approximately 10 Å from the active site causing a displacement of the catalytic aspartate residues. The so- called “first generation” NNI drugs are generally susceptible to the effects of single-point mutations within RT, while more recent “second generation” NNIs, such as efavirenz, the car- boxanilide UC-781 and certain quinoxalines demonstrate much greater resilience to mutations in RT. The crystal structures of the complexes of wild type and mutant RTs with first and second generation NNIs have shown that, for an inhibitor to be potent as well as mutation resilient, it should (i) make hydrogen bonds with the main chain of RT, (ii) have a large number of interactions with RT and (iii) have the ability to rearrange and adapt to a mutated NNI pocket. Based on the crystal structures of the complexes of wild type RT and Tyr188Cys mutant of RT with UC-781, we have designed a small peptide inhibitor. Docking results on this peptide using AutoDock3.0 and SYBYL 6.8.1 indicate that the peptide has a potency comparable to that of UC-781 with a retention of activity against the Tyr188Cys mutant RT. The proposed, small peptide is seen to possess all the desirable features of a potent and mutation resilient inhibitor and is hence a potential lead compound.

Structural basis of Bacillus anthracis MoxXT disruption and the modulation of MoxT ribonuclease activity by rationally designed peptides

Bacillus anthracis MoxXT is a Type II proteic Toxin–Antitoxin (TA) module wherein MoxT is a ribonuclease that cleaves RNA specifically while MoxX interacts with MoxT and inhibits its activity. Disruption of the TA interaction has been proposed as a novel antibacterial strategy. Peptides, either based on antitoxin sequence or rationally designed, have previously been reported to disrupt the MoxXT interaction but cause a decrease in MoxT ribonuclease activity. In the present study, we report the crystal structure of MoxT, and the effect of several peptides in disrupting the MoxXT interaction as well as augmentation of MoxT ribonuclease activity by binding to MoxT in vitro. Docking studies on the peptides were carried out in order to explain the observed structure activity relationships. The peptides with ribonuclease augmentation activity possess a distinct structure and are proposed to bind to a distinct site on MoxT. The docking of the active peptides with MoxT showed that they possess an aromatic group that occupies a conserved hydrophobic pocket. Additionally, the peptides inducing high ribonuclease activity were anchored by a negatively charged group near a cluster of positively charged residues present near the pocket. Our study provides a structural basis and rationale for the observed properties of the peptides and may aid the development of small molecules to disrupt the TA interaction.

Molecular Modeling of the Complex of Endothelin-1(ET-1) with the Endothelin Type A (ET A ) Receptor and the Rational Design of a Peptide Antagonist

Abstract ET-1 is the most potent vasoconstrictor known to date, causing vasoconstriction when bound to the ETa receptor. Inhibitors of the binding of ET-1 to the ETA receptor would be of immense value as potential therapeutic agents in the treatment of cardiovascular disorders such as angina and hypertension. We present here the rational design of such an inhibitor, which is arrived at on the basis of a model of the ET-1/ETA receptor complex proposed by us. The model is found to be consistent with binding and mutagenesis studies of ET-1 as well as of BQ123, a known, potent ETA-selective antagonist which competes with ET-1 for receptor binding. BQ123 is a peptidic antagonist which is constrained to adopt a definite conformation on account of its cyclic nature. The noncyclic peptide antagonist designed by us also has a unique conformation because it contains two dehydro-Alanine (δAla) residues which, on account of their planarity, cause the peptide backbone to bend in a specific and predictable manner. The folding rules for peptides containing δAla were derived in our earlier studies. Energy minimization and modelling of the complex of the designed peptide with the ETA receptor indicate that the antagonist is ETA -selective and the binding is more stable and more specific as compared to that of BQ123.