Description of the topographical changes associated to the different stages of the DsbA catalytic cycle.

This paper provides a description of the surface topography of DsbA, the bacterial disulfide-bond forming enzyme, in the different phases of its catalytic cycle. Three representative states, that is, oxidized and reduced protein and a covalent complex mimicking the DsbA-substrate disulfide intermediate, have been investigated by a combination of limited proteolysis experiments and mass spectrometry methodologies. Protease-accessible sites are largely distributed in the oxidized form with a small predominance inside the thioredoxin domain. Proteolysis occurs even in secondary structure elements, revealing a significant mobility of the protein. Many cleavage sites disappear in the reduced form and most of the remaining ones appear with strongly reduced kinetics. The protein within the complex shows an intermediate behavior. This variation of flexibility in DsbA is probably the determining factor for the course of its catalytic cycle. In particular, the great mobility of the oxidized protein might facilitate the accommodation of its various substrates, whereas the increasing rigidity from the complexed to the reduced form could help the release of oxidized products. The formation of the complex between PID peptide and DsbA does not significantly protect the enzyme against proteolysis, reinforcing the results previously obtained by calorimetry concerning the weakness of their interaction. The few cleavage sites observed, however, are in favor of the presence of the peptide in the binding site postulated from crystallographic studies. As for the peptide itself, the proteolytic pattern and the protection effect exerted by DsbA could be explained by a preferential orientation within the binding site

Des enzymes sur mesure, rêve ou réalité ?

International audienc

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

International audienc

A Microplate Assay for Trypanothione Reductase Inhibitors

International audienc

Preparation of trisulfide derivatives of cystine and their formation as by-products during peptide synthesis

International audienc

Dynamic combinatorial assembly of peptide-rhenium coordinates: Application to the selection of hCyp-18 inhibitors from a library of 12 × 16 components

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Reduction of a Trisulfide Derivative of Glutathione by Glutathione Reductase

International audienc

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