Search

Nucleocytoplasmic shuttling of viral elements, supported by several host factors, is essential for the replication of the human immunodeficiency virus (HIV). HIV‐1 uses a nuclear RNA export pathway mediated by viral protein Rev to transport its Rev response element (RRE)‐containing partially spliced and unspliced transcripts aided by the host nuclear RNA export protein CRM1. The factor(s) interacting with the CRM1‐Rev complex are potential antiretroviral target(s) and could serve as a retroviral model system to study nuclear export machinery adapted by these viruses. We earlier reported that cellular Staufen‐2 interacts with Rev, facilitating viral‐RNA export. Here, we identified the formation of a complex between Staufen‐2, CRM1 and Rev. Molecular docking and simulations mapped the interacting residues in the RNA‐binding Domain 4 of Staufen‐2 as R336 and R337, which were experimentally verified to be critical for interactions among Staufen‐2, CRM1 and Rev by mutational analysis. Staufen‐2 mutants defective in interaction with CRM1 or Rev failed to supplement the Rev‐RNA export activity and viral production, demonstrating the importance of these interactions. Rev‐dependent reporter assays and proviral DNA‐construct transfection‐based studies in Staufen‐2 knockout cells in the presence of leptomycin‐B (LMB) revealed a significant reduction in CRM1‐mediated Rev‐dependent RNA export with decreased virus production as compared to Staufen‐2 knockout background or LMB treatment alone, suggesting the relevance of these interactions in augmenting RNA export activity of Rev. Our observations provide further insights into the mechanistic intricacies of unspliced viral‐RNA export to the cytoplasm and support the notion that abrogating such interactions can reduce HIV‐1 proliferation. [ABSTRACT FROM AUTHOR]

Elucidation of signaling events in a pathogen is potentially important to tackle the infection caused by it. Such events mediated by protein phosphorylation play important roles in infection, and therefore, to predict the phosphosites and substrates of the serine/threonine protein kinases, we have developed a Machine learning‐based approach for Mycobacterium tuberculosis serine/threonine protein kinases using kinase‐peptide structure–sequence data. This approach utilizes features derived from kinase three‐dimensional‐structure environment and known phosphosite sequences to generate support vector machine (SVM)‐based kinase‐specific predictions of phosphosites of serine/threonine protein kinases (STPKs) with no or scarce data of their substrates. SVM outperformed the four machine learning algorithms we tried (random forest, logistic regression, SVM, and k‐nearest neighbors) with an area under the curve receiver‐operating characteristic value of 0.88 on the independent testing dataset and a 10‐fold cross‐validation accuracy of ~81.6% for the final model. Our predicted phosphosites of M. tuberculosis STPKs form a useful resource for experimental biologists enabling elucidation of STPK mediated posttranslational regulation of important cellular processes. [ABSTRACT FROM AUTHOR]

Among the two GroEL paralogs in Mycobacterium tuberculosis, GroEL1 and GroEL2, GroEL1 has a characteristic histidine‐rich C terminus. Since histidine richness is likely to be involved in metal binding, we attempted to decipher the role of GroEL1 in chelating metals and the consequence on M. tuberculosis physiology. Isothermal titration calorimetry showed that GroEL1 binds copper and other metals. Mycobacterial viability assay, redox balance, and DNA protection assay concluded that GroEL1 protects from copper stress in vitro. Solution X‐ray scattering and constrained modeling of GroEL1 −/+ copper ions showed reorientation of the apical domain as seen in functional assembly. We conclude that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and consequent functional divergence of chaperonins. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Diabetes is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The transcription factor Rv0081 of Mycobacterium tuberculosis controls hypoxic gene expression and acts as a regulatory hub in the latent phase of tuberculosis (TB) infection. We report here the crystal structure of Rv0081 at 2.9 Å resolution revealing that it belongs to the well‐known ArsR/SmtB family proteins. However, unlike other members in this family, Rv0081 has neither a metal‐binding domain nor does it possess Cys residues, suggesting an alternate mechanism of gene regulation. Our structural and biochemical analyses suggest the molecular basis for the recognition of self‐regulatory DNA sequences and a plausible mechanism of regulation of Rv0081 in the latent phase of TB infection. Database: Structural data are available in the Protein Data Bank under the accession number – 6JMI [ABSTRACT FROM AUTHOR]

High throughput structural genomics efforts have been making the structures of proteins available even before their function has been fully characterized. Therefore, methods that exploit the structural knowledge to provide evidence about the functions of proteins would be useful. Such methods would beneeded to complement the sequence-based function annotation approaches. The current study describes generation of 3D-structural motifs for metal-binding sites from the known metalloproteins. It then scans all the available protein structures in the PDB database for putative metal-binding sites. Our analysis predicted more than 1000 novel metal-binding sites in proteins using three-residue templates, and more than 150 novel metal-binding sites using four-residue templates. Prediction of metal-binding site in a yeast protein YDR533c led to the hypothesis that it might function as metal-dependent amidopeptidase. The structural motifs identified by our method present novel metal-binding sites that reveal newer mechanisms for a few well-known proteins. Proteins 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The thioredoxin system exists ubiquitously and participates in essential antioxidant and redox-regulation processes via a pair of conserved cysteine residues. In Mycobacterium tuberculosis, which lacks a genuine glutathione system, the thioredoxin system provides reducing equivalents inside the cell. The three-dimensional structure of thioredoxin reductase from M. tuberculosis has been determined at 3 Å resolution. TLS refinement reveals a large libration axis, showing that NADPH-binding domain has large anisotropic disorder. The relative rotation of the NADPH domain with respect to the FAD domain is necessary for the thioredoxin reduction cycle, as it brings the spatially distant reacting sites close together. Normal-mode analysis carried out based on the elastic network model shows that the motion required to bring about the functional conformational change can be accounted for by motion along one single mode. TLS refinement and normal-mode analysis thus enhance our understanding of the associated conformational changes. [ABSTRACT FROM AUTHOR]

Organic solvents are known to bring about dehydration of proteins, the molecular basis of which has remained uncharacterized. The dehydration effect in many cases leads to eventual unfolding of proteins through the macroscopic solvent effect. In some cases, the organic solvent molecules also bind to protein surfaces, thereby forcing local unfolding. The X-ray structure of hen egg-white lysozyme co-crystallized in the presence of alcohols with varying hydrophobicities has been studied. It was noticed that although the alcohols have very little effect on the conformation of the overall protein structure, they profoundly affect protein hydration and disorder of the bound waters. Systematic analysis of the water structure around the lysozyme molecule suggests that an increasing order of hydrophobicity of alcohols is directly proportional to the higher number of weakly bound waters in the protein. As anticipated, the water molecules in the native structure with high temperature factors (≥40 Ų) attain higher disorder in the presence of alcohols. It is believed that the disorder induced in the water molecules is a direct consequence of alcohol binding. [ABSTRACT FROM AUTHOR]

Chaperonin-60 (cpn60) and chaperonin-10 (cpn10) are essential proteins involved in ATP-dependent folding of several intracellular proteins in the bacterial cell. Folding of the nascent substrate polypeptide takes place in the large central cavity formed by each ring of the tetradecameric cpn60. This large cavity is closed upon capping by the heptameric cpn10. Cpn10s interact with cpn60s primarily through a 17-residue mobile loop and regulate the release and binding of the substrate polypeptide from the cpn60 surface. Here, the structure of M. tuberculosis cpn10 is reported at 3.5 Å resolution. The overall structure of the cpn10 monomer is formed of a four-stranded β-barrel and two long stretches of highly flexible segments: the dome loop and the mobile loop. The seven subunits in the heptamer show very little conformational difference and exhibit nearly perfect seven-fold geometry. The binding sites for metal ions in the dome loop of cpn10 have been identified, suggesting the role of metal ions in the stabilization of the protein. Comparisons with the available cpn10 structures indicate several interesting features. [ABSTRACT FROM AUTHOR]

The possible role of the central β-domain (residues 151-287) of streptokinase (SK) was probed by site-specifically altering two charged residues at a time to alanines in a region (residues 230-290) previously identified by Peptide Walking to play a key role in plasminogen (PG) activation. These mutants were then screened for altered ability to activate equimolar 'partner' human PG, or altered interaction with substrate PG resulting in an overall compromised capability for substrate PG processing. Of the eight initial alanine-linker mutants of SK, one mutant, viz. SKKK256, 257aa (SK-D1), showed a roughly 20-fold reduction in PG activator activity in comparison to wild-type SK expressed in Escherichia coli (nSK). Five other mutants were as active as nSK, with two [SKRE248.249aa and SKEK281.282aa, referred to as SK(C) and SK(H), respectively] showing specific activities approximately one-half and two-thirds, respectively, that of nSK. Unlike SK(C) and SK(H), however, SK(D1) showed an extended initial delay in the kinetics of PG activation. These features were drastically accentuated when the charges on the two Lys residues at positions 256 and 257 of nSK were reversed, to obtain SKKK256.257ee [SK(D2)]. This mutant showed a PG activator activity approximately 10-fold less than that of SK(D1). Remarkably, inclusion of small amounts of human plasmin (PN) in the PG activation reactions of SK(D2) resulted in a dramatic, PN dose-dependent rejuvenation of its PG activation capability, indicating that it required pre-existing PN to form a functional activator since it could not effect active site exposure in partner PG on its own, a conclusion further confirmed by its inability to show a 'burst' of p-nitrophenol release in the presence of equimolar human PG and p-nitrophenyl guanidino benzoate. The steady-state kinetic parameters for HPG activation of its 1:1 complex with human PN revealed that although it could form a highly functional activator once 'supplied' with a mature active site, the Km for PG was increased nearly eightfold in comparison to that of nSK-PN. SK mutants carrying simultaneous two- and three-site charge-cluster alterations, viz., SKRE248.249aa;ek281.282aa [SK(CH)], SKEK272.273AA;EK281.282AA [SK(FH)], and SKRE248.249AA;EK272.273AA;EK281.282AA [SK(CFH)], showed additive/synergistic influence of multiple charge-cluster mutations on HPG activation when compared to the respective 'single-site' mutants, with the 'triple-site' mutant [SK(CFH)] showing absolutely no detectable HPG activation ability. Nevertheless, like the other constructs, the double- and triple-charge cluster mutants retained a native like affinity for complexation with partner PG. Their overall structure also, as judged by far-ultraviolet circular dichroism, was closely similar to that of nSK. These results provide the first experimental evidence for a direct assistance by the SK β-domain in the docking and processing of substrate PG by the activator complex, a facet not readily evident probably because of the flexibility of this domain in the recent X-ray crystal structure of the SK-plasmin light chain complex. [ABSTRACT FROM AUTHOR]

CRP/FNR family members are transcription factors that regulate the transcription of many genes in Escherichia coli and other organisms. Mycobacterium tuberculosis H37Rv contains a probable CRP/FNR homologue encoded by the open reading frame Rv3676. The deletion of this gene is known to cause growth defects in cell culture, in bone marrow-derived macrophages and in a mouse model of tuberculosis. The mycobacterial gene Rv3676 shares ∼32% sequence identity with prototype E. coli CRP. The structure of the protein might provide insight into transcriptional regulation in the pathogen by this protein. The M. tuberculosis CRP/FNR transcription regulator was crystallized in space group P212121, with unit-cell parameters a = 54.1, b = 84.6, c = 101.2 Å. The crystal diffracted to a resolution of 2.9 Å. Matthews coefficient and self-rotation function calculations reveal the presence of two monomers in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Chorismate mutase catalyzes the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine in bacteria, fungi and higher plants. The recent re-annotation of the Mycobacterium tuberculosis genome has revealed the presence of a duplicate set of genes coding for chorismate mutase. The mycobacterial gene Rv1885c bears <20% sequence homology to other bacterial chorismate mutases, thus serving as a potential target for the development of inhibitors specific to the pathogen. The M. tuberculosis chorismate mutase was crystallized in space group C2 and the crystals diffracted to a resolution of 2.2 Å . Matthews coefficient and self-rotation function calculations revealed the presence of two monomers in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (H37Rv), the causative agent of the dreaded disease tuberculosis, contains three thioredoxins and a single thioredoxin reductase. Thioredoxin reductase is a member of the pyridine-nucleotide disulfide oxidoreductase family of flavoenzymes. The thioredoxin reductase gene with a His tag at the C-terminus was expressed in Escherichia coli and purified. The dimeric (70 kDa) protein was incubated with 10 mM DTT for 30 min and then crystallized using the hanging-drop vapour-diffusion method in the presence of 15% PEG 3350 and phosphate-citrate buffer pH 5 at room temperature (298 K). A diffraction data set complete to 3 Å resolution has been collected under cryoconditions and the space group was determined to be P41212, with unit-cell parameters a = 107.4, c = 118.2 Å. Matthews coefficient calculations revealed the presence of two monomers in the asymmetric unit. [ABSTRACT FROM AUTHOR]

The article presents a case study of a 69-year-old woman with vomiting and intermittent right upper quadrant pain. The patient underwent ultrasound scan, which revealed multiple gallstones in a thick walled gallbladder. She also underwernt other tests such as an X-ray, computed tomography (CT) scan of the abdomen, and magnetic resonance cholangiopancreatography (MRCP). A diagnosis of Bouveret's syndrome was made, which usually needs surgical or endoscopic treatment.