The diphtheria toxin repressor (DtxR) fromCorynebacterium diphtheriue is a divalent-metal activated repressor of chromosomal genes responsible for siderophore-mediated ironuptake and of a gene on several corynebacteriophages that encodes diphtheria toxin. Even though DtxR is the best characterized irondependent repressor to date, numerous key properties of the protein still remain to be explained. One is the role of the cation-anion pair discovered in its first metal-binding site. A second is the reason why zinc exhibits its activating effect only at a concentration 100-fold higher than other divalent cations.In the presently reported 1.85 8, resolution Co-DtxR structure at 100K, the sulfate anion in the cation-anion-binding site interacts with three side chains that are all conserved in the entire DtxR family, which points to a possible physiological role of the anion.A comparison of the 1.85 8, Cobalt-DtxR structure at lOOK and the 2.4 8, Zinc-DtxR structure at room temperature revealed no significant differences. Hence, the difference in efficiency of Co2+ and Zn2+ to activate DtxR remains a mystery and might be hidden in the propexties of the intriguing second metal-binding site. Our studies do, however, provide a high resolution view of the cationanion-binding site that has most likely evolved to interact not only with a cation but also with the anion in a very precise manner.Keywords: Corynebacterium diphtheriue; diphtheria toxin repressor; metal-activated repressor; X-ray crystal structure All pathogenic bacteria encounter iron-deficient growth conditions in vivo as most of the iron in the host is sequestered by iron-and
With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2’,6’-dihydroxy-4’-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2’,4’,6’- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.
Summary Leucocyte recruitment is critical during many acute and chronic inflammatory diseases. Chemokines are key mediators of leucocyte recruitment during the inflammatory response, by signalling through specific chemokine G‐protein‐coupled receptors (GPCRs). In addition, chemokines interact with cell‐surface glycosaminoglycans (GAGs) to generate a chemotactic gradient. The chemokine interleukin‐8/CXCL8, a prototypical neutrophil chemoattractant, is characterized by a long, highly positively charged GAG‐binding C‐terminal region, absent in most other chemokines. To examine whether the CXCL8 C‐terminal peptide has a modulatory role in GAG binding during neutrophil recruitment, we synthesized the wild‐type CXCL8 C‐terminal [CXCL8 (54–72)] (Peptide 1), a peptide with a substitution of glutamic acid (E) 70 with lysine (K) (Peptide 2) to increase positive charge; and also, a scrambled sequence peptide (Peptide 3). Surface plasmon resonance showed that Peptide 1, corresponding to the core CXCL8 GAG‐binding region, binds to GAG but Peptide 2 binding was detected at lower concentrations. In the absence of cellular GAG, the peptides did not affect CXCL8‐induced calcium signalling or neutrophil chemotaxis along a diffusion gradient, suggesting no effect on GPCR binding. All peptides equally inhibited neutrophil adhesion to endothelial cells under physiological flow conditions. Peptide 2, with its greater positive charge and binding to polyanionic GAG, inhibited CXCL8‐induced neutrophil transendothelial migration. Our studies suggest that the E70K CXCL8 peptide, may serve as a lead molecule for further development of therapeutic inhibitors of neutrophil‐mediated inflammation based on modulation of chemokine–GAG binding.
The crystal structures of Emycin E (1), di-o-bromobenzoyl-Emycin F (2) and o-bromobenzoyl-Emycin D (3) have been determined by X-ray analysis at low temperature. Emycin E and o-bromobenzoyl-Emycin D both crystallize with two molecules in a triclinic unit cell. These two structures can be solved and refined either in the centrosymmetric space group P1;, with apparent disorder localized at or around the expected chiral centre, or in the non-centrosymmetric space group P1 as mixtures of two diastereomers without disorder. Only the latter interpretation is consistent with the chemical and spectroscopic evidence. Refinements in the centrosymmetric and non-centrosymmetric space groups are compared in this paper and are shown to favour the chemically correct interpretation, more decisively so in the case of the bromo derivative as a result of the anomalous dispersion of bromine. Structures (1) and (3) provide a dramatic warning of the dangers inherent in the conventional wisdom that if a structure can be refined satisfactorarily in both centrosymmetric and non-centrosymmetric space groups, the former should always be chosen. In these two cases, despite apparently acceptable intensity statistics and R factors (5.87 and 3.55%), the choice of the centrosymmetric space group leads to the serious chemical error that the triclinic unit cell contains a racemate rather than two chiral diastereomers! The weakest reflections are shown to be most sensitive to the correct choice of space group, underlining the importance of refining against all data rather than against intensities greater than a specified threshold. The use of similar-distance restraints is shown to be beneficial in both P1 refinements. Di-o-bromobenzoyl-Emycin F crystallizes in the monoclinic space group P2(1) with one molecule in the asymmetric unit and so does not give rise to these problems of interpretation. The absolute configuration of the two bromo derivatives, and hence the Emycins in general, was determined unambiguously as S at the chiral centre C3.
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