DsbC activation by the N-terminal domain of DsbD

Goldstone, David C.; Haebel, Peter; Katzen, Federico; Bader, Martin W.; Bardwell, James C.A.; Beckwith, Jon; Metcalf, Peter

doi:10.1073/pnas.171315498

Cited by 37 publications

(48 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3 and data not shown). This indicates that electrons can be directly donated from the ␣ domain to DsbG, as it has been previously reported for DsbC (13).…”

Section: Resultssupporting

confidence: 81%

See 1 more Smart Citation

Reconstitution of a Disulfide Isomerization System

Collet¹,

Riemer²,

Bader³

et al. 2002

Journal of Biological Chemistry

Self Cite

101

100

View full text Add to dashboard Cite

Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides. In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins. To be active as isomerases, DsbC and DsbG must be kept reduced. This task is performed by the cytoplasmic membrane protein DsbD. DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains. The ␤ domain is membrane-embedded, whereas the ␣ and ␥ domains are localized to the periplasm. It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains. To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the ␣, ␤, ␥, and ␤␥ domains. Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG. We showed that electrons are transferred from thioredoxin to the ␤ domain then successively to the ␥ domain, the ␣ domain, and finally on to DsbC or DsbG. We also determined the redox potential of the ␥ domain to be ؊241 mV, and that of the ␣ domain was found to be ؊229 mV. This shows that the direction of electron flow within DsbD is thermodynamically driven.A critical step in the folding of newly synthesized proteins is the formation of native disulfide bonds between the thiol groups of two cysteine residues (for a review, see Ref. 1). In prokaryotes, this disulfide formation occurs in the periplasm and is catalyzed by a protein called DsbA 1 (DsbA stands for disulfide bond) (2). DsbA is a powerful (3) and rather nonspecific oxidant that has the dangerous potential of introducing non-native disulfides into proteins with multiple cysteines, an event that, if uncorrected, can lead to protein inactivation. To correct for these mistakes, the cell needs a disulfide isomerization system. The isomerization system in Escherichia coli involves three additional Dsb proteins: DsbC, DsbG, and DsbD.DsbC and DsbG are periplasmically localized proteins that have disulfide isomerase activity in vitro. The three-dimensional structure of DsbC solved recently by McCarthy et al. (4) showed that DsbC is a V-shaped dimer of two thioredoxin-like folds. The active site CXXC motifs, one from each monomer, face into the interior of the V. It appears that dimerization of DsbC is required for its isomerase activity (5).To be able to correct non-native disulfides, the cysteines of the CXXC motif within DsbC and DsbG need to be kept reduced in the oxidizing environment of the periplasm. In the isomerization reaction, the first cysteine of the CXXC motif, present in the reduced form, is thought to attack a non-native disulfide in a misfolded protein. This results in the formation of a mixeddisulfide between the isomerase and the target protein. This mixed disulfide is resolved either by the attack of the secon...

show abstract

“…3 and data not shown). This indicates that electrons can be directly donated from the ␣ domain to DsbG, as it has been previously reported for DsbC (13).…”

Section: Resultssupporting

confidence: 81%

“…This suggests that electrons are successively transferred from thioredoxin to the ␤ domain, to the ␥ and ␣ domains, and finally on to DsbC or DsbG. In addition, in vitro 4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonate trapping experiments also suggested that the ␣ domain and DsbC interact (13).…”

mentioning

confidence: 99%

Reconstitution of a Disulfide Isomerization System

Collet¹,

Riemer²,

Bader³

et al. 2002

Journal of Biological Chemistry

Self Cite

101

100

View full text Add to dashboard Cite

show abstract

“…Also, Bader et al (8) suggest that dimerization protects the DsbC active site from the interaction with DsbB, thus maintaining the segregation of the oxidative and reductive pathways. The recent characterization of the DsbC-DsbD␣ complex crystal structure revealed that the dimerization domain of DsbC is critical for the interaction with DsbD and is, therefore, required for reduction to take place (9). Nonetheless, interactions between enzymes within the same pathway are favored strongly over nonphysiological disulfide-exchange reactions between the two pathways by kinetic differences of 10 3 -to 10 7 -fold (10).…”

mentioning

confidence: 99%

Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli : Reconciling two competing pathways

Segatori

Paukstelis

Gilbert

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

In the Escherichia coli periplasm, the formation of protein disulfide bonds is catalyzed by DsbA and DsbC. DsbA is a monomer that is maintained in a fully oxidized state by the membrane enzyme DsbB, whereas DsbC is a dimer that is kept reduced by a second membrane protein, DsbD. Although the catalytic regions of DsbA and DsbC are composed of structurally homologous thioredoxin motif domains, DsbA serves only as an oxidase in vivo, whereas DsbC catalyzes disulfide reduction and isomerization and also exhibits significant chaperone activity. To reconcile the distinct catalytic activities of DsbC and DsbA, we constructed a series of chimeras comprising of the dimerization domain of DsbC, with or without the adjacent ␣-helical linker region, fused either to the first, second, third, or fifth residue of intact DsbA or to thioredoxin. The chimeras fully substituted for DsbC in disulfide-bond rearrangement and also were able to restore protein oxidation in a dsbA background. Remarkably, the chimeras could serve as a single catalyst for both disulfide-bond formation and rearrangement, thus reconciling the kinetically competing DsbBDsbA and DsbD-DsbC pathways. This property appeared to depend on the orientation of the DsbA active-site cysteines with respect to the DsbC dimerization domain. In vitro, the chimeras had high chaperone activity and significant reductase activity but only 15-22% of the disulfide-isomerization activity of DsbC, suggesting that rearrangement of nonnative disulfides may be mediated primarily by cycles of random reduction and reoxidation. D isulfide bonds play a key role in the folding process of many secretory and membrane proteins. A series of cysteine thiol:disulfide oxidoreductases have evolved in both eukaryotic and prokaryotic systems to catalyze this critical cellular process. In particular, the Dsb family of the Escherichia coli periplasm consists of two distinct pathways: DsbA-DsbB and DsbC͞DsbG-DsbD. These pathways are involved in the formation of disulfides and the rearrangement of incorrectly formed bonds, respectively (1, 2). The extreme oxidizing nature of DsbA mediates rapid oxidation of substrate cysteines and, therefore, results in the formation of nonnative disulfides, which are in turn rearranged by DsbC and, to a lesser extent, DsbG. Despite the strong oxidizing environment of the periplasmic space, DsbC and DsbG have to be maintained in a reduced state to be able to catalyze the rearrangement of nonnative disulfide bonds (1, 2).DsbA and DsbC are found in entirely oxidized and reduced states, respectively (3). DsbA is recycled by the membrane protein DsbB, which then passes its electrons to the quinones and the respiratory chain, whereas DsbC is maintained in the reduced state by another membrane protein, DsbD, by means of an electrontransfer relay that involves thioredoxin (TrxA) and TrxA reductase in the cytoplasm (4). Remarkably, the Dsb machinery has evolved to capitalize on a strong thiol oxidant (DsbA) and a strong thiol reductant (DsbC) that do not appear to exchange ele...

show abstract

“…In vitro, DsbA is a potent catalyst of protein cysteine oxidation, whereas DsbC exhibits disulfide isomerase activity. The membrane proteins DsbB and DsbD appear to be responsible for maintaining DsbA and DsbC, respectively, in the proper oxidative state for optimal function (Goldstone et al, 2001;Ito & Inaba, 2008). In the present study, K2S was modified to contain 357 of the 527 amino acids of the human tPA and remained nine disulfide bonds, which challenged the active production in E. coli.…”

Section: Discussionmentioning

confidence: 78%