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

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

Modeling of CCR5 Recognition by HIV-1 gp120: How the Viral Protein Exploits the Conformational Plasticity of the Coreceptor

International audienceThe chemokine receptor CCR5 is a key player in HIV-1 infection. The cryo-EM 3D structure of HIV-1 envelope glycoprotein (Env) subunit gp120 in complex with CD4 and CCR5 has provided important structural insights into HIV-1/host cell interaction, yet it has not explained the signaling properties of Env nor the fact that CCR5 exists in distinct forms that show distinct Env binding properties. We used classical molecular dynamics and site-directed mutagenesis to characterize the CCR5 conformations stabilized by four gp120s, from laboratory-adapted and primary HIV-1 strains, and which were previously shown to bind differentially to distinct CCR5 forms and to exhibit distinct cellular tropisms. The comparative analysis of the simulated structures reveals that the different gp120s do indeed stabilize CCR5 in different conformational ensembles. They differentially reorient extracellular loops 2 and 3 of CCR5 and thus accessibility to the transmembrane binding cavity. They also reshape this cavity differently and give rise to different positions of intracellular ends of transmembrane helices 5, 6 and 7 of the receptor and of its third intracellular loop, which may in turn influence the G protein binding region differently. These results suggest that the binding of gp120s to CCR5 may have different functional outcomes, which could result in different properties for viruses

Local Interaction Density (LID), a Fast and Efficient Tool to Prioritize Docking Poses

International audienceLigand docking at a protein site can be improved by prioritizing poses by similarity to validated binding modes found in the crystal structures of ligand/protein complexes. The interactions formed in the predicted model are searched in each of the reference 3D structures, taken individually. We propose to merge the information provided by all references, creating a single representation of all known binding modes. The method is called LID, an acronym for Local Interaction Density. LID was benchmarked in a pose prediction exercise on 19 proteins and 1382 ligands using PLANTS as docking software. It was also tested in a virtual screening challenge on eight proteins, with a dataset of 140,000 compounds from DUD-E and PubChem. LID significantly improved the performance of the docking program in both pose prediction and virtual screening. The gain is comparable to that obtained with a rescoring approach based on the individual comparison of reference binding modes (the GRIM method). Importantly, LID is effective with a small number of references. LID calculation time is negligible compared to the docking time