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

Abstract

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...

Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

“…Protein expression and purification was performed as previously described (29). All proteins used in this study were more than 95% pure as judged by Coomassie Blue-stained SDS-PAGE electrophoresis.…”

“…SF100 ⌬degP ⌬dsbC cells were grown in LB medium and SF100 ⌬degP dsbA::kan cells in minimal medium (M9 salts, 0.1% casein amino acids, 2 mM MgSO 4 , 5 g/ml thiamine, 0.4% glycerol with 50 g/ml of kanamycin) supplemented with 25 g/ml of chloramphenicol and 100 g/ml of ampicillin at 37°C. Induction of protein synthesis, cell harvesting, and assays for tPA activity were performed as described earlier (29). For the evaluation of in vivo oxidase activity of DsbC and its variants, E. coli LM106 (MC1000 dsbA::kan) and LM102 (MC1000 dsbB::kan) were transformed with the appropriate pBAD33 derivatives, grown in LB medium with 50 g/ml of kanamycin and 25 g/ml of chloramphenicol, and subcultured 1:50 in low phosphate minimal medium (MOPS salts, 0.2% glycerol, 0.2% casein amino acids, and 0.5 g/ml thiamine, with 50 g/ml of kanamycin and 25 g/ml of chloramphenicol).…”

“…mDsbC-mDsbC exhibited slightly lower activity in the oxidative refolding of reduced RNase A and in the reduction of insulin (84 and 78%, respectively) relative to wt DsbC; however these differences in activity were not statistically significant. The in vivo disulfide isomerase activity of mDsbC-mDsbC in the E. coli periplasm was evaluated by determining the yield of correctly folded and active vtPA secreted via the StII signal peptide (29). vtPA is a truncated version of human tPA containing 9 disulfide bonds.…”

“…Moreover, unlike PDI, the significance of the putative peptide binding cleft of DsbC on disulfide isomerization has not been ascertained. However, while DsbA or TrxA with a PDI active site dipeptide (CGHC) display very little isomerase activity in vitro and in vivo (27)(28)(29), we recently showed that upon fusion to a dimerization region that provides a putative substrate binding surface (the E. coli peptidyl proline isomerase FkpA) they acquire the ability to assist the folding of periplasmically expressed multidisulfide heterologous proteins (30).…”

Engineering antibody fragments to fold in the absence of disulfide bonds

“…Protein expression and purification were performed by IMAC and size exclusion chromatography as previously described. 41 All proteins used in this study were more than 90% pure as judged by Coomassie Brilliant Blue-stained SDS-PAGE. Cell pellets were resolubilized in 5Â SDS-PAGE loading buffer to give total protein fractions.…”