The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

Abstract

The assembly of the ␤-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded ␤-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 ␤-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential ␤-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only ␤-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 ␤-barrel proteins. The outer membrane (OM)4 is a complex macromolecular structure that is essential for the viability of Gram-negative bacteria and serves as a permeability barrier against hydrophobic antibiotics. It is a unique asymmetric lipid bilayer with phospholipids forming the inner leaflet and lipopolysaccharides (LPS) forming the outer leaflet. The proteins that are present in the OM are either lipoproteins that are anchored to the inner leaflet via a lipid moiety or ␤-barrel proteins that are integral membrane proteins. The mechanisms that govern the assembly of the OM are poorly understood (1-3). In particular, we have a poor understanding of how the ␤-barrel proteins, which are synthesized in the cytoplasm and translocated unfolded across the inner membrane by the Sec translocon, are then 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). Two partially redundant periplasmic chaperone pathways have been described in the Escherichia coli periplasm (5, 6). The first one involves SurA, a periplasmic prolyl cistrans-isomerase, which is considered as the primary chaperone in the periplasm. SurA has been shown to be involved in the biogenesis of the most abundant ␤-barrel proteins, such as OmpA, OmpF, and OmpC (6, 7), and is the preferred chaperone for the essential protein LptD (previously Imp) (8). The second pathway involves the chaperone Skp and the protease/ chaperone protein DegP. This pathway has been proposed to serve as a backup for SurA, assisting the biogenesis of the proteins that fell off the SurA pathway (6). The fact that surAskp and surAdegP mutants are not viable (5) indicates that cells cannot survive without at least o...

The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli

“…The association of LptD with its partner, LptE (4), and/or the isomerase activity of DsbC (15), rearranges this disulfide such that Cys 173 now becomes bonded to the fourth cysteine (Cys 725 ) to make the 2-4 disulfide bond. At this point, DsbA reengages LptD to catalyze a second disulfide bond between the remaining cysteines (Cys 31 and Cys 724 ), resulting in the 1-3 disulfide bond (16).…”

Crystal Structure of the Outer Membrane Protein RcsF, a New Substrate for the Periplasmic Protein-disulfide Isomerase DsbC

“…These reducing equivalents maintain DsbC in a reduced state in the periplasm, allowing the protein to function as an isomerase/reductase (see below). Several DsbC substrates with multiple cysteines have already been identified; they include four periplasmic enzymes (42,43) as well as the essential ␤-barrel protein LptD (44). The presence of two disulfides formed between cysteines that are not consecutive in the sequence of RcsF suggested that the assembly of this protein involves DsbC.…”

Protease homolog BepA (YfgC) promotes assembly and degradation of β-barrel membrane proteins in Escherichia coli

“…Analysis with a DsbA mutant that forms kinetically stable mixed-disulfide intermediates with substrates suggested that DsbA is involved in formation of both nonnative and native disulfide bonds in LptD (17). A previous study also suggested that DsbC participates in the oxidative folding of LptD (17,20) although disruption of the dsbC gene had little effect on the stability of LptD (19).…”