Biomedical subjects

Esther Kellenberger

Publications and source records attributed to Esther Kellenberger.

10 recordsLinked to original sources

PubMed2006

Redesign of Schistosoma mansoni NAD+ catabolizing enzyme: active site H103W mutation restores ADP-ribosyl cyclase activity.

Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes that are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change, we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products (i.e., a value larger than observed for other members of this family such as CD38). Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provides new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge, however, in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR.

ADP-ribosyl Cyclase↗

PubMed2006

A chemogenomic analysis of the transmembrane binding cavity of human G-protein-coupled receptors.

The amino acid sequences of 369 human nonolfactory G-protein-coupled receptors (GPCRs) have been aligned at the seven transmembrane domain (TM) and used to extract the nature of 30 critical residues supposed--from the X-ray structure of bovine rhodopsin bound to retinal--to line the TM binding cavity of ground-state receptors. Interestingly, the clustering of human GPCRs from these 30 residues mirrors the recently described phylogenetic tree of full-sequence human GPCRs (Fredriksson et al., Mol Pharmacol 2003;63:1256-1272) with few exceptions. A TM cavity could be found for all investigated GPCRs with physicochemical properties matching that of their cognate ligands. The current approach allows a very fast comparison of most human GPCRs from the focused perspective of the predicted TM cavity and permits to easily detect key residues that drive ligand selectivity or promiscuity.

Amino Acid Sequence↗

PubMed2005

Production of calcium-mobilizing metabolites by a novel member of the ADP-ribosyl cyclase family expressed in Schistosoma mansoni.

ADP-ribosyl cyclases are structurally conserved enzymes that are best known for catalyzing the production of the calcium-mobilizing metabolite, cyclic adenosine diphosphate ribose (cADPR), from nicotinamide adenine dinucleotide (NAD(+)). However, these enzymes also produce adenosine diphosphate ribose (ADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP(+)), both of which have been shown to modulate calcium mobilization in vitro. We have now characterized a new member of the cyclase family from Schistosoma mansoni, a member of the Platyhelminthes phylum. We show that the novel NAD(P)(+) catabolizing enzyme (NACE) expressed by schistosomes is structurally most closely related to the cyclases cloned from Aplysia but also shows significant homology with the mammalian cyclases, CD38 and CD157. NACE expression is developmentally regulated in schistosomes, and the GPI-anchored protein is localized to the outer tegument of the adult schistosome. Importantly, NACE, like all members of the cyclase family, is a multifunctional enzyme and catalyzes NAD(+) glycohydrolase and base-exchange reactions to produce ADPR and NAADP(+). However, despite being competent to generate a cyclic product from NGD(+), a nonphysiologic surrogate substrate, NACE is so far the only enzyme in the cyclase family that is unable to produce significant amounts of cADPR (<0.02% of reaction products) using NAD(+) as the substrate. This suggests that the other calcium-mobilizing metabolites produced by NACE may be more important for calcium signaling in schistosomes. Alternatively, the function of NACE may be to catabolize extracellular NAD(+) to prevent its use by host enzymes that utilize this source of NAD(+) to facilitate immune responses.

ADP-ribosyl Cyclase↗

PubMed2005

Dynamics and metal exchange properties of C4C4 RING domains from CNOT4 and the p44 subunit of TFIIH.

Zinc fingers are small structured protein domains that require the coordination of zinc for a stable tertiary fold. Together with FYVE and PHD, the RING domain forms a distinct class of zinc-binding domains, where two zinc ions are ligated in a cross-braced manner, with the first and third pairs of ligands coordinating one zinc ion, while the second and fourth pairs ligate the other zinc ion. To investigate the relationship between the stability and dynamic behaviour of the domains and the stability of the metal-binding site, we studied metal exchange for the C4C4 RING domains of CNOT4 and the p44 subunit of TFIIH. We found that Zn(2+)-Cd(2+) exchange is different between the two metal-binding sites in the C4C4 RING domains of the two proteins. In order to understand the origins of these distinct exchange rates, we studied the backbone dynamics of both domains in the presence of zinc and of cadmium by NMR spectroscopy. The differential stability of the two metal-binding sites in the RING domains, as reflected by the different metal exchange rates, can be explained by a combination of accessibility and an electrostatic ion interaction model. A greater backbone flexibility for the p44 RING domain as compared to CNOT4 may be related to the distinct types of protein-protein interactions in which the two C4C4 RING domains are involved.

Amino Acid Sequence↗

PubMed2005

Solution structure of the C-terminal domain of TFIIH P44 subunit reveals a novel type of C4C4 ring domain involved in protein-protein interactions.

The human general transcription factor TFIIH is involved in both transcription and DNA nucleotide excision repair. Among the 10 subunits of the complex, p44 subunit plays a crucial role in both mechanisms. Its N-terminal domain interacts with the XPD helicase, whereas its C-terminal domain is involved specifically in the promoter escape activity. By mutating an exposed and non-conserved cysteine residue into a serine, we produced a soluble mutant of p44-(321-395) suitable for solution structure determination. The domain adopts a C4C4 RING domain structure with sequential organization of beta-strands that is related to canonical RING domains by a circular permutation of the beta-sheet elements. Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321-395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, thus, differs from previously reported interaction mechanisms involving RING domains.

Amino Acid Sequence↗

PubMed2004

Comparative evaluation of eight docking tools for docking and virtual screening accuracy.

Eight docking programs (DOCK, FLEXX, FRED, GLIDE, GOLD, SLIDE, SURFLEX, and QXP) that can be used for either single-ligand docking or database screening have been compared for their propensity to recover the X-ray pose of 100 small-molecular-weight ligands, and for their capacity to discriminate known inhibitors of an enzyme (thymidine kinase) from randomly chosen "drug-like" molecules. Interestingly, both properties are found to be correlated, since the tools showing the best docking accuracy (GLIDE, GOLD, and SURFLEX) are also the most successful in ranking known inhibitors in a virtual screening experiment. Moreover, the current study pinpoints some physicochemical descriptors of either the ligand or its cognate protein-binding site that generally lead to docking/scoring inaccuracies.

Computer Graphics↗

PubMed2004

Structure of the His44 --> Ala single point mutant of the distal finger motif of HIV-1 nucleocapsid protein: a combined NMR, molecular dynamics simulation, and fluorescence study.

The nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers that strongly bind Zn(2+) through coordination of one His and three Cys residues. It has been suggested that NCp7 function is conformation specific since substitution of any of the zinc coordinating residues in the zinc finger motifs leads to subsequent loss of viral infectivity. To further determine the structural requirements necessary for this specific conformation, we investigated by (1)H 2D NMR and molecular dynamics simulations the structure of the distal finger motif of NCp7 in which the zinc coordinating amino acid, His 44, was substituted by a noncoordinating Ala residue. While the fold of the N-terminal part of this mutated peptide was similar to that of the native peptide, an increased lability and significant conformational changes were observed in the vicinity of the His-to-Ala mutation. Moreover, molecular dynamics simulations suggested a mechanism by which the variant peptide can bind zinc ion even though one zinc-coordinating amino acid was lacking. Using the fluorescence of the naturally occurring Trp37 residue, the binding affinity of the variant peptide to the (TG)(3) model oligonucleotide was found to be decreased by about 2 orders of magnitude with respect with the native peptide. Modeling of the DNA:NCp7 complex using structures of the variant peptide suggests that the residues forming a hydrophobic cleft in the native protein are improperly oriented for efficient DNA binding by the variant peptide.

Alanine↗

PubMed2004

Recovering the true targets of specific ligands by virtual screening of the protein data bank.

The Protein Data Bank (PDB) has been processed to extract a screening protein library (sc-PDB) of 2148 entries. A knowledge-based detection algorithm has been applied to 18,000 PDB files to find regular expressions corresponding to either protein, ions, co-factors, solvent, or ligand atoms. The sc-PDB database comprises high-resolution X-ray structures of proteins for which (i) a well-defined active site exists, (ii) the bound-ligand is a small molecular weight molecule. The database has been screened by an inverse docking tool derived from the GOLD program to recover the known target of four unrelated ligands. Both the database and the inverse screening procedures are accurate enough to rank the true target of the four investigated ligands among the top 1% scorers, with 70-100 fold enrichment with respect to random screening. Applying the proposed screening procedure to a small-sized generic ligand was much less accurate suggesting that inverse screening shall be reserved to rather selective compounds.

Algorithms↗

PubMed2002

Induced folding of the U2AF35 RRM upon binding to U2AF65.

The human essential splicing factor U2AF (U2 auxiliary factor) consists of 35 and 65 kDa subunits which form a highly stable heterodimer in solution. Copurification of the recombinant U2AF35 RNA recognition motif (U2AF35 RRM) and full-length U2AF65 yields a soluble and functionally active minimal U2AF heterodimer. Recombinant U2AF35 RRM protein free and in complex with three different regions of U2AF65 was characterized by nuclear magnetic resonance spectroscopy. We found that the recombinant U2AF35 RRM is unstructured in solution but its tertiary structure is induced upon binding to U2AF65. This interaction is mediated by the N-terminal proline-rich region of U2AF65 and does not involve the U2AF65 RRMs.

Amino Acid Motifs↗

PubMedDate not supplied

sc-PDB: an annotated database of druggable binding sites from the Protein Data Bank.

The sc-PDB is a collection of 6 415 three-dimensional structures of binding sites found in the Protein Data Bank (PDB). Binding sites were extracted from all high-resolution crystal structures in which a complex between a protein cavity and a small-molecular-weight ligand could be identified. Importantly, ligands are considered from a pharmacological and not a structural point of view. Therefore, solvents, detergents, and most metal ions are not stored in the sc-PDB. Ligands are classified into four main categories: nucleotides (< 4-mer), peptides (< 9-mer), cofactors, and organic compounds. The corresponding binding site is formed by all protein residues (including amino acids, cofactors, and important metal ions) with at least one atom within 6.5 angstroms of any ligand atom. The database was carefully annotated by browsing several protein databases (PDB, UniProt, and GO) and storing, for every sc-PDB entry, the following features: protein name, function, source, domain and mutations, ligand name, and structure. The repository of ligands has also been archived by diversity analysis of molecular scaffolds, and several chemoinformatics descriptors were computed to better understand the chemical space covered by stored ligands. The sc-PDB may be used for several purposes: (i) screening a collection of binding sites for predicting the most likely target(s) of any ligand, (ii) analyzing the molecular similarity between different cavities, and (iii) deriving rules that describe the relationship between ligand pharmacophoric points and active-site properties. The database is periodically updated and accessible on the web at http://bioinfo-pharma.u-strasbg.fr/scPDB/.

Algorithms↗