Abstract
The assembly of the β-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded β-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of β-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential β-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only β-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important β-barrel proteins.
Highlights
Translocon, are transported across the periplasm and inserted in the outer membrane (OM)
The first cysteine of the CXXC catalytic motif of DsbC performs a nucleophilic attack on a non-native disulfide present in a substrate protein, which results in the formation of an unstable mixed disulfide complex between DsbC and the substrate
The surAdsbC Mutants Have a Synthetic Phenotype—We set out to determine whether DsbC is involved in the mechanisms that govern OM biogenesis
Summary
Translocon, are transported across the periplasm and inserted in the OM. According to the most widely accepted model, unfolded -barrel proteins are escorted across the periplasm by soluble periplasmic chaperones that deliver them to an OM multiprotein complex involving the -barrel protein BamA (3, 4). One of the proteins displaying a chaperone activity in vitro is DsbC, a protein-disulfide isomerase involved in the oxidative folding of periplasmic proteins with multiple cysteine residues (9, 10). The first cysteine of the CXXC catalytic motif of DsbC performs a nucleophilic attack on a non-native disulfide present in a substrate protein, which results in the formation of an unstable mixed disulfide complex between DsbC and the substrate. This mixed disulfide will be resolved either by attack of another cysteine of the misfolded protein, resulting in the formation of a more stable disulfide in the substrate and the release of reduced DsbC, or by attack of the other cysteine of the CXXC motif In this latter case, DsbC functions as a reductase, reducing the non-native disulfide and giving DsbA a new chance to form a correct disulfide. The active site cysteine residues are not required for this chaperone activity (19)
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