Characterization of Determinants for the Specificity of Arabidopsis Thioredoxins h in Yeast Complementation

International audienceThe disruption of the two thioredoxin genes in Sac-charomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H 2 O 2 tolerance, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce me-thionine sulfoxide assimilation and restore a normal cell cycle. In addition AtTRX2 also confers growth on sulfate but no H 2 O 2 tolerance. In contrast, AtTRX3 does not confer growth on sulfate but induces H 2 O 2 tolerance. We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H 2 O 2 tolerance is located in the N-terminal part of AtTRX3. In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipo-tent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins. After duplications each member evolved by losing or masking some of the determinants

Discovery of Fragment Molecules That Bind the Human Peroxiredoxin 5 Active Site

The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors

Première étude structurale et dynamique d'une peroxyrédoxine par RMN

Les cellules luttent en permanence contre les pro-oxydants et leurs effets néfastes. Les hydroperoxydes en sont les plus courants. La cystéine nucléophile du site actif confère aux peroxyrédoxines (Prx) une activité peroxydasique, réductrice des hydroperoxydes dépendante notamment de thiorédoxines. Pour la première fois, une Prx, Ahp1 de Saccharomyces cerevisiae, a fait l'objet d'une étude structurale par RMN triple résonance (1H/13C/15N) sur un échantillon uniformément marqué [13C/15N/50 % 2H] en milieu aqueux réducteur. Sa topologie est semblable à celle des autres Prx. L'analyse des données de relaxation RMN 15N a émontré que Ahp1 est un homodimère 2x19,4kDa en solution, ce qui a été confirmé par ultracentrifugation analytique et par des calculs d'hydrodynamique. La forme suroxydée, préparée in vitro par un excès de tBuOOH, a été cractérisée grâce à un marquage sélectif Cys[b-13C]. Les résultats montrent que la cystéine du site actif est alors oxydée en sulfonateLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Études conformationnelles par RMN d'antibiotiques antifongiques à mode d'action stérol-dépendant, et de nouvelles toxines animales

La spectroscopie RMN a été utilisée pour résoudre la structure tridimensionnelle de molécules biologiques en solution possédant une activité bien établie. Tout d'abord, une étude conformationnelle a été effectuée pour la bacillomycine L et les plipastatines A et B dans le DMSO, deux lipopeptides cycliques synthétisés par Bacillus subtilis qui possèdent une activité antifongique stérol-dépendante. Les plipastatines ainsi que la surfactine, un autre lipopeptide produit par B. subtilis possédant des propriétés surfactantes importantes, ont ensuite été marquées biosynthétiquement à l'15N. La conformation de ces deux composés a ainsi été étudiée en interaction avec des micelles de SDS dans l'eau et il a été observé, que contrairement aux plipastatines, la surfactine montrait une structure assez similaire dans le DMSO et dans cet environnement micellaire. Dans le chapitre suivant, nous avons étudié la conformation de 6 macrolides polyéniques représentatifs, tous issus du genre Streptomyces. Cette étude nous a permis de dégager des propriétés communes en solutions qui seront des informations utiles pour l'interprétation des relations structure/activité de ces composés. Enfin, dans le dernier chapitre, nous avons élucidé la structure tridimensionnelle de deux nouvelles toxines agissant sur le système neuromusculaire, la conotoxine EVIA et la sarafotoxine-m issus respectivement du venin d'un cône marin (Conus ermineus) et d'un serpent africain (Atractaspis microlepidota microlepidota). Des informations capitales pour les relations structure/activité de ces composés ont été révélées. En particulier, pour la conotoxine EVIA, un phénomène d'isomérisation cis/trans au niveau d'une liaison peptidique X-Pro s'est avérée apparemment essentiel pour la reconnaissance avec son récepteur neuronal canal Na+.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

A test of parafoveal-on-foveal effects with pairs of orthographically related words

One of the main controversies in the field of eye movements in reading concerns the question of whether the processing of two adjacent words in reading occurs in sequence, or in parallel. To distinguish between these views, the present experiment tested the presence of parafoveal-on-foveal effects with pairs of orthographically related words (or neighbours that differed by a single letter) in a controlled but reading-like situation. Results revealed that fixation times on a foveal target word were shorter when the target was accompanied by an orthographically similar parafoveal word than when the parafoveal word was dissimilar. Furthermore, the size of the effect tended to vary with both the relative frequency of target and parafoveal words, and the position of the critical letter. These results were interpreted in the framework of a pure parallel processing hypothesis, where the processing of adjacent words is only limited by visual acuity, and the respective lexical properties of the foveal and the parafoveal words

NMR structure of antibiotics plipastatins A and B from Bacillus subtilis inhibitors of phospholipase A2

AbstractPlipastatins A and B are antifungal antibiotics belonging to a family of lipopeptides capable of inhibiting phospholipase A2 (PLA2) and are biosynthesised under certain circumstances by Bacillus subtilis. U-15N plipastatins A and B were obtained from cultures of the strain NCIB 8872 on a Landy medium modified for stable-isotope labelling by the substitution of the L-glutamic acid used as the sole nitrogen source, by 15NH4Cl. These two lipo-decapeptides, lactonised by esterification of the Ile10 C-terminus with the phenolic hydroxyl of Tyr3, differ only by a D-Ala (plipastatin A)/D-Val (plipastatin B) substitution at the position 6. The 1H- and 15N-nuclear magnetic resonance (NMR) signals of a 4:6 mixture of plipastatins A and B were unambiguously assigned and their structures in dimethylsulfoxide solution were calculated on the basis of a set of NMR-derived restraints. Plipastatins A and B are well-defined structures in solution stabilised by a type 1 β-turn comprising residues 6–9 and several other specific hydrogen bonds. The structures afford a first molecular basis for the future studies of their biological activities both in lipidic layers or on PLA2

Binding evaluation of fragment-based scaffolds for probing allosteric enzymes

International audienceFragment-based drug discovery has become a powerful method for the generation of drug leads against therapeutic targets. Beyond the identification of novel and effective starting points for drug design, fragments have emerged as reliable tools for assessing protein druggability and identifying protein hot spots. Here, we have examined fragments resulting from the deconstruction of known inhibitors from the glycogen phosphorylase enzyme, a therapeutic target against type 2 diabetes, with two motivations. First, we have analyzed the fragment binding to the multiple binding sites of the glycogen phosphorylase, and then we have investigated the use of fragments to study allosteric enzymes. The work we report illustrates the power of fragmentlike ligands not only for probing the various binding pockets of proteins, but also for uncovering cooperativity between these various binding sites

Stereochemical studies on the polyene macrolide nystatin A1: the hydroxyl groups in the C-1—C-10 fragment are all-syn

International audienceTwo monoacetylated fragments 1 and 2 corresponding to the C-1—C-10 portion of nystatin A1 have been prepared by controlled degradation. The 3R, 5R and 7R relative configurations at the asymmetric centers of these fragments were determined by 2D-proton phase-sensitive DQF-COSY nmr and nOe difference spectroscopy.The 3R*, 5R* and 7R* relative configurations at the asymmetric centers of C-1–C-10 fragments of nystatin A. were determined by 2D-proton phase-sensitive bQF-COSY nmr and nOe difference spectroscopy

MD exploration of human peroxiredoxin V interactions with ligands

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Dynamical properties of the loop 320s of substrate-free and substrate-bound muscle creatine kinase by NMR

International audienceMuscle creatine kinase (MCK; ) is a 86 kDa homodimer that belongs to the family of guanidino kinases. MCK has been intensively studied for several decades, but it is still not known why it is a dimer because this quaternary structure does not translate into obvious structural or functional advantages over the homologous monomeric arginine kinase. In particular, it remains to be demonstrated whether MCK subunits are independent. Here, we describe NMR chemical-shift perturbation and relaxation experiments designed to study the active site 320s flexible loop of this enzyme. The analysis was performed with the enzyme in its ligand-free and MgADP-complexed forms, as well as with the transition-state analogue abortive complex (MCKMgADPcreatinenitrate ion). Our data indicate that each subunit can bind substrates independently