Biomedical subjects

E Quéméneur

Publications and source records attributed to E Quéméneur.

14 recordsLinked to original sources

PubMed2006

Structural insights into protein-uranyl interaction: towards an in silico detection method.

Documenting the modes of interaction of uranyl (UO(2)2+) with large biomolecules, and particularly with proteins, is instrumental for the interpretation of its behavior in vitro and in vivo. The gathering of three-dimensional information concerning uranyl-first shell atoms from two structural databases, the Cambridge Structural Databank and the Protein Data Bank (PDB) allowed a screening of corresponding topologies in proteins of known structure. In the computer-aided procedure, all potentially bound residues from the template structure were granted full flexibility using a rotamer library. The Amber force-field was used to loosen constraints and score each predicted site. Our algorithm was validated as a first stage through the recognition of existing experimental data in the PDB. The coherent localization of missing atoms in the density map of an ambiguous uranium/uranyl-protein complex exemplified the efficiency of our approach, which is currently suggesting the experimental investigation of uranyl-protein binding site.

Computers↗

PubMed2000

Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex.

Approaching the molecular mechanism of some enzymes is hindered by the difficulty of obtaining suitable protein-ligand complexes for structural characterization. DsbA, the major disulfide oxidase in the bacterial periplasm, is such an enzyme. Its structure has been well characterized in both its oxidized and its reduced states, but structural data about DsbA-peptide complexes are still missing. We report herein an original, straightforward, and versatile strategy for making a stable covalent complex with a cysteine-homoalanine thioether bond instead of the labile cystine disulfide bond which normally forms between the enzyme and polypeptides during the catalytic cycle of DsbA. We substituted a bromohomoalanine for the cysteine in a model 14-mer peptide derived from DsbB (PID-Br), the membrane partner of DsbA. When incubated in the presence of the enzyme, a selective nucleophilic substitution of the bromine by the thiolate of the DsbA Cys(30) occurred. The major advantage of this strategy is that it enables the direct use of the wild-type form of the enzyme, which is the most relevant to obtain unbiased information on the enzymatic mechanism. Numerous intermolecular NOEs between DsbA and PID could be observed by NMR, indicating the presence of preferential noncovalent interactions between the two partners. The thermodynamic properties of the DsbA-PID complex were measured by differential scanning calorimetry. In the complex, the values for both denaturation temperature and variation in enthalpy associated with thermal unfolding were between those of oxidized and reduced forms of DsbA. This progressive increase in stability along the DsbA catalytic pathway strongly supports the model of a thermodynamically driven mechanism.

Alkylation↗

PubMed2000

Design of a Gag pentapeptide analogue that binds human cyclophilin A more efficiently than the entire capsid protein: new insights for the development of novel anti-HIV-1 drugs.

Cyclophilin A (hCyp-18), a ubiquitous cytoplasmic peptidyl-prolyl cis/trans isomerase (PPIase), orchestrates HIV-1 core packaging. hCyp-18, incorporated into the virion, enables core uncoating and RNA release and consequently plays a critical role in the viral replication process. hCyp-18 specifically interacts with a single exposed loop of the Gag polyprotein capsid domain via a network of nine hydrogen bonds which mainly implicates a 7-mer fragment of the loop. As previously reported, the corresponding linear heptapeptide Ac-Val-His-Ala-Gly-Pro-Ile-Ala-NH(2) (2) binds to hCyp-18 with a low affinity (IC(50) = 850 +/- 220 microM) but a potentially useful selectivity for hCyp-18 relative to hFKBP-12, another abundant PPIase. On the basis of X-ray structures of Gag fragments:hCyp-18 complexes, we generated a series of modified peptides in order to probe the determinants of the interaction and hence to select a peptidic ligand displaying a higher affinity than the capsid domain of Gag. We synthesized a series of heptapeptides to test the energetic contribution of amino acids besides the Gly-Pro moiety. In particular the importance of the histidine residue for the interaction was underscored. We also investigated the influence of N- and C-terminal modifications. Hexapeptides containing either deaminovaline (Dav) in place of the N-terminal valine or substitution of the C-terminal alanine amide with a benzylamide group displayed increased affinities. Combination of both modifications gave the most potent competitor Dav-His-Ala-Gly-Pro-Ile-NHBn (28) which has a higher affinity for hCyp-18 (K(d) = 3 +/- 0.5 microM) than the entire capsid protein (K(d) = 16 +/- 4 microM) and a very low affinity for hFKBP-12. Some of our results strongly suggest that the title compound is not a substrate of hCyp-18 and interacts preferentially in the trans conformation.

Amino Acid Sequence↗

PubMed2000

Tailoring new enzyme functions by rational redesign.

Site-directed mutagenesis is still a very efficient strategy to elaborate improved enzymes. Recently, advances have been made in developing rational strategies aimed at reshaping enzyme specificities and mechanisms, and at engineering biocatalysts through molecular assembling. These knowledge-based studies greatly benefit from the most recent computational analyses of enzyme structures and functions. The combination of rational and combinatorial methods opens up new vistas in the design of stable and efficient enzymes.

Animals↗

PubMed1999

On the non-respect of the thermodynamic cycle by DsbA variants.

The mechanism of the disulfide-bond forming enzyme DsbA depends on the very low pKa of a cysteine residue in its active-site and on the relative instability of the oxidized enzyme compared to the reduced one. A thermodynamic cycle has been used to correlate its redox properties to the difference in the free energies of folding (deltadeltaGred/ox) of the oxidized and reduced forms. However, the relation was proved unsatisfied for a number of DsbA variants. In this study, we investigate the thermodynamic and redox properties of a highly destabilized variant DsbA(P151A) (substitution of cis-Pro151 by an alanine) by the means of intrinsic tryptophan fluorescence and by high-sensitivity differential scanning calorimetry (HS-DSC). When the value of deltadeltaGred/ox obtained fluorimetrically for DsbA(P151A) does not correlate with the value expected from its redox potential, the value of deltadeltaGred/ox provided by HS-DSC are in perfect agreement with the predicted thermodynamic cycle for both wild-type and variant. HS-DSC data indicate that oxidized wild-type enzyme and the reduced forms of both wild-type and variant unfold according to a two-state mechanism. Oxidized DsbA(P151A) shows a deviation from two-state behavior that implies the loss of interdomain cooperativity in DsbA caused by Pro151 substitution. The presence of chaotrope in fluorimetric measurements could facilitate domain uncoupling so that the fluorescence probe (Trp76) does not reflect the whole unfolding process of DsbA(P151A) anymore. Thus, theoretical thermodynamic cycle is respected when an appropriate method is applied to DsbA unfolding under conditions in which protein domains still conserve their cooperativity.

Calorimetry, Differential Scanning↗

PubMed1999

On the role of the cis-proline residue in the active site of DsbA.

In addition to the Cys-Xaa-Xaa-Cys motif at position 30-33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis-proline in proximity of the active site residues. In the variant DsbA(P151A), this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild-type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbA(P151A) crystallized in a different crystal form from the wild-type protein, in space group P2(1) with six molecules in the asymmetric unit. Its X-ray structure was determined to 2.8 A resolution. The most significant conformational changes occurred at the active site. The loop 149-152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys-Xaa-Xaa-Cys sequence presented in four out of six molecules in the asymmetric unit a gauche conformation not observed in the wild-type protein. The X-ray structure and folding studies on DsbA(P151A) were consistent with the cis-proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.

Bacterial Proteins↗

PubMed1998

The length of a single turn controls the overall folding rate of "three-fingered" snake toxins.

Snake curaremimetic toxins are short all-beta proteins, containing several disulfide bonds which largely contribute to their stability. The four disulfides present in snake toxins make a "disulfide beta-cross"-fold that was suggested to be a good protein folding template. Previous studies on the refolding of snake toxins (Ménez, A. et al. (1980) Biochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggested that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and close to the "disulfide beta-cross". To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxins, toxin alpha (Naja nigricollis) and erabutoxin b (Laticauda semifasciata), and two synthetic homologues, the alpha mutants, alpha60 and alpha62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one residue in the turn length that reproduced the natural heterogeneity at that locus. The refolding was studied by electrospray mass spectrometry (ESMS) time-course analysis. This analysis permitted both the identification and quantitation of the population of intermediates present during the process. All toxins were shown to share the same sequential scheme for disulfide bond formation despite large differences in their refolding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed differences in the refolding rate of toxins.

Alkylation↗

PubMed1998

Engineering cyclophilin into a proline-specific endopeptidase.

Designing an enzyme requires, among a number of parameters, the appropriate positioning of catalytic machinery within a substrate-binding cleft. Using the structures of cyclophilin-peptide complexes, we have engineered a new catalytic activity into an Escherichia coli cyclophilin by mutating three amino acids, close to the peptide binding cleft, to form a catalytic triad similar to that found in serine proteases. In conjunction with cyclophilin's specificity for proline-bearing peptides, this creates a unique endopeptidase, cyproase 1, which cleaves peptides on the amino-side of proline residues. When acting on an Ala-Pro dipeptide, cyproase 1 has an efficiency (kcat/Km) of 0.7 x 10(4) M(-1) s(-1) and enhances the rate of reaction (kcat/kuncat) 8 x 10(8)-fold. This activity depends upon a deprotonated histidine and is inhibited by nucleophile-specific reagents, as occurs in natural serine proteases. Cyproase 1 can hydrolyse a protein substrate with a proline-specific endoprotease activity.

Catalysis↗

PubMed1998

Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy.

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13C(alpha) 13CO, 1H(alpha), and 13Cbeta NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple (1H-13C-15N) and double resonance (1H-15N or 1H-13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3(J)H(N)H(alpha) and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro 151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13C(alpha) chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.

Bacterial Proteins↗

PubMed1997

Glutathione-dependent activities of Trypanosoma cruzi p52 makes it a new member of the thiol:disulphide oxidoreductase family.

Trypanothione: glutathione disulphide thioltransferase of Try-panosoma cruzi (p52) is a key enzyme in the regulation of the intracellular thiol-disulphide redox balance by reducing glutathione disulphide. Here we show that p52, like other disulphide oxidoreductases possessing the CXXC active site motif, catalyses the reduction of low-molecular-mass disulphides (hydroxyethyl-disulphide) as well as protein disulphides (insulin). However, p52 seems to be a poor oxidase under physiological conditions as evidenced by its very low rate for oxidative renaturation of reduced ribonuclease A Like thioltransferase and protein disulphide isomerase, p52 was found to possess a glutathione-dependent dehydroascorbate reductase activity. The kinetic parameters were in the same range as those determined for mammalian dehydroascorbate reductases. A catalytic mechanism taking into account both trypanothione- and glutathione-dependent reduction reactions was proposed. This newly characterized enzyme is specific for the parasite and provides a new target for specific chemotherapy.

Animals↗

PubMed1996

ATP binding and hydrolysis by the multifunctional protein disulfide isomerase.

We previously reported the ability of protein disulfide isomerase (PDI) to undergo an ATP-dependent autophosphorylation. Our efforts to map the modification site have been hindered by the low abundance and instability of the labeling. Results are presented in this paper on the nature of phospho-PDI, which appears as an intermediate with a half-life of 2.5-8.8 min in an ATPase reaction. ATP binds to PDI with high affinity, Kd 9.66 microM, and the kinetic parameters KmATP and kcat of the ATPase reaction were measured by using a pyruvate kinase-lactate dehydrogenase-coupled assay under various conditions. Strikingly, the ATPase reaction is stimulated in the presence of denatured polypeptides, while the disulfide oxidization activity of PDI is not affected by ATP. However, PDI is known to participate in various unrelated functions in the endoplasmic reticulum, and ATP could be involved in the regulation of one of these. The results are discussed in light of recent findings on ATP-chaperone relationships.

Adenosine Triphosphatases↗

PubMed1995

Increased sensitivity of autoradiography and fluorography by membrane blotting.

The detection of low amounts of 3H-, 14C- or 35S-labeled compounds can be difficult with x-ray films. Here we report the benefits of blotting labeled molecules that have been electrophoretically separated to a membrane prior to autoradiographic or fluorographic detection. Blotting allows a reduction, by a factor of 5-10, in exposure times compared with dried gels. When combined with a rapid and safe alternative to the impregnation of blots with liquid scintillant, a further 3-fold to 5-fold increase in sensitivity is achieved. Bands containing as low as 1 Bq 14C or 100 to 10(3) Bq 3H could be detected after 24 hours of exposure. This enhancement in sensitivity may lead to new applications of 3H and 14C.

Autoradiography↗

PubMed1994

A major phosphoprotein of the endoplasmic reticulum is protein disulfide isomerase.

One of the effects of ATP in the endoplasmic reticulum is to induce the phosphorylation of several proteins among which a 57-kDa protein (pp57) prevails in our labeling conditions. We provide evidence that pp57 is protein disulfide isomerase (PDI), an abundant ubiquitous protein of the endoplasmic reticulum involved in various important cellular functions. This phosphorylation does not result from the activity of a microsomal protein kinase but from an autophosphorylation as described for other microsomal proteins such as chaperones. Phosphoamino acid analysis and cyanogen bromide cleavage indicate that the modification site lies on a threonine residue within the central region of the protein outside the thioredoxin-like domains. For the pure PDI, only the dimer is able to phosphorylate, while some experiments suggest that within the endoplasmic reticulum the phosphorylated form of PDI is mainly mobilized in larger size oligomers. Thus a possible role for this phosphorylation may be to modulate the association of PDI with its different partners.

Amino Acid Sequence↗

PubMed1994

A pleîotropic acid phosphatase-deficient mutant of Escherichia coli shows premature termination in the dsbA gene. Use of dsbA::phoA fusions to localize a structurally important domain in DsbA.

A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phosphatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the periplasmic space was isolated. The mutation which mapped close to chlB, at 87 min on the E. coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed from TnphoA fusions to genes encoding periplasmic or membrane proteins. A DNA fragment that complements the mutation was cloned and shown to carry the dsbA gene, which encodes a periplasmic disulphide bond-forming factor. The mutant had an ochre triplet in dsbA, truncating the protein at amino acid 70. Introduction of TnphoA fusions into a plasmid-borne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all exported to the periplasmic space in both dsbA+ and dsbA strains. They belong to three different classes, depending on the length of the DsbA fragment fused to PhoA. When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA+ strain, as in the case of wild-type PhoA. Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retained both the DsbA and PhoA activities. Those with shorter DsbA fragments that still carried the -Cys-Pro-His-Cys- motif were rapidly degraded (no DsbA activity). The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential for its folding into a proteolytic-resistant and enzymatically active form.

Acid Phosphatase↗