Evidence that the pathway of disulfide bond formation in Escherichia coli involves interactions between the cysteines of DsbB and DsbA.
Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. Chem. 270, 17072-17074] have described similar findings.) We isolated a dominant negative mutant of dsbA, dsbAd, where Cys-33 of the DsbA active site is changed to tyrosine. Both DsbAd and DsbA are able to form a mixed disulfide with DsbB, which may be an intermediate in the reoxidation of DsbA. This complex is more stable with DsbAd. The dominance can be suppressed by increasing the production of DsbB. By using mutants of DsbB in which one or two cysteines have been changed to alanine, we show that only Cys-104 is important for complex formation. Therefore, we suggest that in vivo, reduced DsbA forms a complex with DsbB in which Cys-30 of DsbA is disulfide-bonded to Cys-104 of DsbB. Cys-104 is rapidly replaced by Cys-33 of DsbA to generate the oxidized form of this protein.
Guilhot
C
C
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Jander
G
G
Martin
N L
NL
Beckwith
J
J
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001710:Biotin; D015153:Blotting, Western; D002458:Cell Fractionation; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005799:Genes, Dominant; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D011994:Recombinant Proteins; D001710:Biotin; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine