Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism

DETERMINATION PAR RESONANCE MAGNETIQUE NUCLEAIRE DE LA STRUCTURE TRIDIMENSIONNELLE DE PETITES PROTEINES (_ PEPTIDE CHIMERE STABILISE PAR DES PONTS DISULFURE _ DOMAINE CARBOXY-TERMINAL DE LA SOUS-UNITE P44 DU FACTEUR DE TRANSCRIPTION HUMAIN TFIIH)

LA PREMIERE PARTIE TRAITE D'INGENIERIE DES PROTEINES. UNE SEQUENCE DE TYPE RGD A ETE INTRODUITE DANS UNE TOXINE DE SCORPION CONTENANT TROIS PONTS DISULFURE. LA TOXINE CHIMERE POSSEDE DES PROPRIETES ANTIAGREGANTES. SA STRUCTURE TRIDIMENSIONNELLE REVELE QU'UNE HELICE ALPHA PEUT MIMER UN COUDE BETA DE TYPE II. UNE DEUXIEME PARTIE S'INTERESSE AUX PROTEINES QUI LIENT LE ZINC ET QUI SONT IMPLIQUEES DANS LA TRANSCRIPTION DES GENES DE CLASSES II CHEZ LES EUCARYOTES. _ UNE REVUE BIBLIOGRAPHIQUE DECRIT LES PROPRIETES DU ZINC ET LES DIFFERENTS MOTIFS STRUCTURAUX DE LIAISON AU ZINC. _ UN CHAPITRE EST CONSACRE A LA PURIFICATION DE LA SOUS-UNITE HRPB10ALPHA DE L'ARN POLYMERASE II HUMAINE. CETTE SOUS-UNITE DE 58 ACIDES AMINES LIE UN ION ZN 2 +. LE COMPORTEMENT BIOCHIMIQUE AINSI QUE LES PREMIERS SPECTRES ENREGISTRES SUR LA PROTEINE PURIFIEE METTENT EN EVIDENCE UNE TENDANCE A L'AGREGATION. _ UN CHAPITRE PRESENTE LA DETERMINATION DE LA STRUCTURE TRIDIMENSIONNELLE DU DOMAINE CARBOXY-TERMINAL DE LA SOUS-UNITE P44 (P44 3 2 1 - 3 9 5) DE TFIIH. TFIIH EST UN FACTEUR DE BASE DE LA TRANSCRIPTION. IL EST AUSSI UN ACTEUR ESSENTIEL DE LA REPARATION DE L'ADN PAR EXCISION DE NUCLEOTIDES. LE DOMAINE P44 3 2 1 - 3 9 5 LIE DEUX IONS ZN 2 +. L'ETUDE STRUCTURALE REVELE UN FEUILLET BETA ANTIPARALLELE A TROIS BRINS ASSOCIE A UNE HELICE ALPHA. ELLE TEMOIGNE EGALEMENT D'UNE DYNAMIQUE LOCALISEE AUTOUR DE L'UN DES SITES DE LIAISON AU ZINC. LE MOTIF DE COORDINATION DES IONS ZINC DE P44 3 2 1 - 3 9 5 EST ORIGINAL, BIEN QUE LA TOPOLOGIE DU DOMAINE SOIT COMMUN A D'AUTRES PROTEINES DE LIAISON AU ZINC. LE TRAVAIL SUR P44 A FAIT APPEL A DEUX TECHNIQUES PROPRES A LA RESONANCE MAGNETIQUE NUCLEAIRE DES MACROMOLECULES BIOLOGIQUES : (1) LE MARQUAGE ISOTOPIQUE ET (2) L'AUTOMATISATION DE L'ATTRIBUTION DES PICS DE CORRELATION NOES. L'UTILISATION ET L'EVALUATION DE CES TECHNIQUES SONT DETAILLEES DANS UNE PARTIE METHODOLOGIE.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Genotypage et essais cliniques

STRASBOURG ILLKIRCH-Pharmacie (672182101) / SudocSudocFranceF

sc-PDB: a database for identifying variations and multiplicity of 'druggable' binding sites in proteins.

International audienceBACKGROUND: The sc-PDB database is an annotated archive of druggable binding sites extracted from the Protein Data Bank. It contains all-atoms coordinates for 8166 protein-ligand complexes, chosen for their geometrical and physico-chemical properties. The sc-PDB provides a functional annotation for proteins, a chemical description for ligands and the detailed intermolecular interactions for complexes. The sc-PDB now includes a hierarchical classification of all the binding sites within a functional class. METHOD: The sc-PDB entries were first clustered according to the protein name indifferent of the species. For each cluster, we identified dissimilar sites (e.g. catalytic and allosteric sites of an enzyme). SCOPE AND APPLICATIONS: The classification of sc-PDB targets by binding site diversity was intended to facilitate chemogenomics approaches to drug design. In ligand-based approaches, it avoids comparing ligands that do not share the same binding site. In structure-based approaches, it permits to quantitatively evaluate the diversity of the binding site definition (variations in size, sequence and/or structure). AVAILABILITY: The sc-PDB database is freely available at: http://bioinfo-pharma.u-strasbg.fr/scPDB

sc-PDB: a database for identifying variations and multiplicity of 'druggable' binding sites in proteins.

International audienceBACKGROUND: The sc-PDB database is an annotated archive of druggable binding sites extracted from the Protein Data Bank. It contains all-atoms coordinates for 8166 protein-ligand complexes, chosen for their geometrical and physico-chemical properties. The sc-PDB provides a functional annotation for proteins, a chemical description for ligands and the detailed intermolecular interactions for complexes. The sc-PDB now includes a hierarchical classification of all the binding sites within a functional class. METHOD: The sc-PDB entries were first clustered according to the protein name indifferent of the species. For each cluster, we identified dissimilar sites (e.g. catalytic and allosteric sites of an enzyme). SCOPE AND APPLICATIONS: The classification of sc-PDB targets by binding site diversity was intended to facilitate chemogenomics approaches to drug design. In ligand-based approaches, it avoids comparing ligands that do not share the same binding site. In structure-based approaches, it permits to quantitatively evaluate the diversity of the binding site definition (variations in size, sequence and/or structure). AVAILABILITY: The sc-PDB database is freely available at: http://bioinfo-pharma.u-strasbg.fr/scPDB

Modeling the allosteric modulation of CCR5 function by Maraviroc

International audienceMaraviroc is a non-peptidic, low molecular weight CC chemokine receptor 5 (CCR5) ligand that has recently been marketed for the treatment of HIV infected individuals. This review discusses recent molecular modeling studies of CCR5 by homology to CXC che- mokine receptor 4, their contribution to the under- standing of the allosteric mode of action of the inhibitor and their potential for the development of future drugs with improved efficiency and preservation of CCR5 biological functions

A simple and fuzzy method to align and compare druggable ligand-binding sites.

International audienceA novel method to measure distances between druggable protein cavities is presented. Starting from user-defined ligand binding sites, eight topological and physicochemical properties are projected from cavity-lining protein residues to an 80 triangle-discretised sphere placed at the centre of the binding site, thus defining a cavity fingerprint. Representing binding site properties onto a discretised sphere presents many advantages: (i) a normalised distance between binding sites of different sizes may be easily derived by summing up the normalised differences between the 8 computed descriptors; (ii) a structural alignment of two proteins is simply done by systematically rotating/translating one mobile sphere around one immobile reference; (iii) a certain degree of fuzziness in the comparison is reached by projecting global amino acid properties (e.g., charge, size, functional groups count, distance to the site centre) independently of local rotameric/tautomeric states of cavity-lining residues. The method was implemented in a new program (SiteAlign) and tested in a number of various scenarios: measuring the distance between 376 related active site pairs, computing the cross-similarity of members of a protein family, predicting the targets of ligands with various promiscuity levels. The proposed method is robust enough to detect local similarity among active sites of different sizes, to discriminate between protein subfamilies and to recover the known targets of promiscuous ligands by virtual screening