Etudes fonctionnelles et structurales de la protéine EED, partenaire cellulaire du virus VIH-1 et de la cellulase « froide » Cel5G de Pseudoalteromonas haloplanktis

Thèse réalisée dans le Laboratoire de BioCristallographie du Dr R. HASER et dans le Laboratoire de Virologie et Pathogenèse Virale du Pr. P. BOULANGERThe human protein EED belongs to the Polycomb Group. It acts as a transcriptional repressor and as a gene silencer. EED seems also to be important during the HIV-1 replication cycle, participating in the trafficking of preintegration complex and favouring the IN-mediated DNA integration reaction. EED has been overexpressed and purified in order to be crystallized alone or in complex with its viral partners IN, MA and Nef. A 3D structure model of EED has been obtained by homology modelling. Interaction regions determined by phage-display are localized on loops of the model.The psychrophilic P. haloplanktis produces the cellulase Cel5G. Its crystallographic structure was solved alone and in complex with cellobiose by molecular replacement at 1.4 Å and 1.6 Å resolution, respectively. Cellulase Cel5A from the mesophilic E. chrysanthemi has been used as a search model. Structural details and comparisons give new insights into the understanding of aspects governing the cold adaptation of the enzyme at a molecular level.La protéine humaine EED appartient à la famille des «Polycomb». Elle intervient dans la régulation génique. Elle jouerait aussi un rôle dans le cycle du virus de l'immunodéficience humaine (VIH-1) en participant au transport du complexe de préintégration et en facilitant la réaction d'intégration. EED a été surproduite afin d'être cristallisée seule et en complexe avec les protéines virales IN, MA et Nef. Un modèle de EED a été construit par homologie structurale. D'après nos expériences de « phage display », les régions susceptibles d'interagir avec les partenaires viraux se situent sur des boucles de ce modèle.La bactérie psychrophile P. haloplanktis produit la cellulase Cel5G. Les structures de cette cellulase seule et en complexe avec le cellobiose, ont été déterminées à haute résolution par remplacement moléculaire. La cellulase Cel5A de la bactérie mésophile E. chrysanthemi a été utilisée comme modèle. Nos résultats permettent de mieux comprendre les déterminants structuraux responsables de l'adaptation des enzymes aux basses températures

Études fonctionnelles et structurales de la protéine EED, partenaire cellulaire du virus VIH-1 et de la cellulase "froide" Cel5G de Pseudoalteromonas haloplanktis

La protéine humaine EED appartient à la famille des "Polycomb". Elle intervient dans la régulation génique. Elle jouerait aussi un rôle dans le cycle du virus de l'immunodéficience humaine (VIH-1) en participant au transport du complexe de préintégration et en facilitant la réaction d'intégration. EED a été surproduite afin d'être cristallisée seule et en complexe avec les protéines virales IN, MA et Nef. Un modèle de EED a été construit par homologie structurale. D'après nos expériences de " phage display ", les régions susceptibles d'interagir avec les partenaires viraux se situent sur des boucles de ce modèle. La bactérie psychrophile P. haloplanktis produit la cellulase Cel5G. Les structures de cette cellulase seule et en complexe avec le cellobiose, ont été déterminées à haute résolution par remplacement moléculaire. La cellulase Cel5A de la bactérie mésophile E. chrysanthemi a été utilisée comme modèle. Nos résultats permettent de mieux comprendre les déterminants structuraux responsables de l'adaptation des enzymes aux basses températuresLYON1-BU.Sciences (692662101) / SudocCACHAN-ENS (940162301) / SudocSudocFranceF

Bourgogne, Nièvre, Entrains-sur-Nohain, 8 rue du 8 mai 1945 : construction d'une extension de pavillon individuel : rapport de diagnostic

L’intervention s’est révélée totalement négative documentant par là la limite septentrionale de l'agglomération d’Intaranum

GMP Synthetase: Allostery, Structure, and Function

Glutamine amidotransferases (GATs) catalyze the hydrolysis of glutamine and transfer the generated ammonia to diverse metabolites. The two catalytic activities, glutaminolysis and the subsequent amination of the acceptor substrate, happen in two distinct catalytic pockets connected by a channel that facilitates the movement of ammonia. The de novo pathway for the synthesis of guanosine monophosphate (GMP) from xanthosine monophosphate (XMP) is enabled by the GAT GMP synthetase (GMPS). In most available crystal structures of GATs, the ammonia channel is evident in their native state or upon ligand binding, providing molecular details of the conduit. In addition, conformational changes that enable the coordination of the two catalytic chemistries are also informed by the available structures. In contrast, despite the first structure of a GMPS being published in 1996, the understanding of catalysis in the acceptor domain and inter-domain crosstalk became possible only after the structure of a glutamine-bound mutant of Plasmodium falciparum GMPS was determined. In this review, we present the current status of our understanding of the molecular basis of catalysis in GMPS, becoming the first comprehensive assessment of the biochemical function of this intriguing enzyme

Bourgogne, Nièvre, Entrains-sur-Nohain, 8 rue du 8 mai 1945 : construction d'une extension de pavillon individuel : rapport de diagnostic

L’intervention s’est révélée totalement négative documentant par là la limite septentrionale de l'agglomération d’Intaranum

The Candida glabrata glycogen branching enzyme structure reveals unique features of branching enzymes of the Saccharomycetaceae phylum

International audienceAbstract Branching enzymes (BE) are responsible for the formation of branching points at the 1,6 position in glycogen and starch, by catalyzing the cleavage of α-1,4-linkages and the subsequent transfer by introducing α-1,6-linked glucose branched points. BEs are found in the large GH13 family, eukaryotic BEs being mainly classified in the GH13_8 subfamily, GH13_9 grouping almost exclusively prokaryotic enzymes. With the aim of contributing to the understanding of the mode of recognition and action of the enzymes belonging to GH13_8, and to the understanding of features distinguishing these enzymes from those belonging to subfamily 13_9 we solved the crystal structure of the glycogen branching enzyme (GBE) from the yeast Candida glabrata, CgGBE, in ligand free forms and in complex with a maltotriose. The structures revealed the presence of a domain already observed in Homo sapiens and Oryza sativa BEs and that we named α-helical N-terminal domain, in addition to the three conserved domains found in BE. We confirmed by phylogenetic analysis that this α-helical N-terminal domain is always present in the GH13_8 enzymes suggesting that it could actually present a signature for this subfamily. We identified two binding sites (BS) in the α-helical N-terminal domain and in the carbohydrate binding module 48 (CBM48), respectively, which show a unique structural organization only present in the Saccharomycotina phylum. Our structural and phylogenetic investigation provides new insight into the structural characterization of GH13_8 GBE revealing unique structural features only present in the Saccharomycotina phylum thereby conferring original properties to this group of enzymes

Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED-2

crystals and analysis by SDS-PAGE and Coomassie blue staining. Lane 1 : solution of purified EED3-H(10 mg/mL) used for crystallogenesis (protein load : 50 μg). Lane 2 : protein content of solubilized single crystal. MW : markers of protein molecular mass, indicated in kilodaltons (kDa) on the right side of the panel.<p><b>Copyright information:</b></p><p>Taken from "Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED"</p><p>http://www.virologyj.com/content/5/1/32</p><p>Virology Journal 2008;5():32-32.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292171.</p><p></p

Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED-0

crystals and analysis by SDS-PAGE and Coomassie blue staining. Lane 1 : solution of purified EED3-H(10 mg/mL) used for crystallogenesis (protein load : 50 μg). Lane 2 : protein content of solubilized single crystal. MW : markers of protein molecular mass, indicated in kilodaltons (kDa) on the right side of the panel.<p><b>Copyright information:</b></p><p>Taken from "Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED"</p><p>http://www.virologyj.com/content/5/1/32</p><p>Virology Journal 2008;5():32-32.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292171.</p><p></p

Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED-1

. Shown is a ribbon representation of the polypeptide backbone atoms of EED3 isoform (amino acid residues 84–441), with secondary and tertiary structures of the different β-blades. , 3D-model of the EED3 seven-bladed β-propeller, deduced from crystallographic data (modified, from [30]). The black arrow indicates the major difference between our putative model (A) and the crystal model (B), consisting of the α1 helical region facing the β-strand β17 in β-blade IV. , Position of immunogenic epitopes (depicted in green) on the 3D-model of EED polypeptide backbone (represented in blue). , Primary and secondary structures of EED3, deduced from crystallographic data [30]. The amino acid sequence was numbered according to the accepted nomenclature [12] : Met95 in EED1 isoform represented Met1 in EED3 ; thus, the C-terminal residue L440 in EED3 corresponded to L535 in EED1. Regions in β-strand structure are represented by horizontal arrows, with reference to the blade number and β-strand letter a, b, c or d ; α-helices are represented by spirals, and turns by TT. Helical regions marked α1 and η1, and the β-strand region marked β17, were structurized domains of EED which were unique among representatives of WD-40 proteins. The relative accessibility of each residue () in the 3D structure was extracted from the dictionary of protein structure [45], and indicated as coloured bars under the sequence with the following colour code : dark blue, highly accessible ; light blue, accessible ; white, buried. Discrete regions recognized by anti-EED IgG are indicated by green boxes. The binding sites of HIV-1 matrix protein (MA) and integrase (IN) are underlined by solid black lines.<p><b>Copyright information:</b></p><p>Taken from "Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED"</p><p>http://www.virologyj.com/content/5/1/32</p><p>Virology Journal 2008;5():32-32.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292171.</p><p></p