An Engineered Pathway for the Formation of Protein Disulfide Bonds

In Gram-negative bacteria, the introduction of disulfide bonds into folding proteins occurs in the periplasm and is catalyzed by donation of an energetically unstable disulfide from DsbA, which is subsequently re-oxidized through interaction with DsbB. Gram-positive bacteria lack a classic periplasm but nonetheless encode Dsb-like proteins. Staphylococcus aureus encodes just one Dsb protein, a DsbA, and no DsbB. Here we report the crystal structure of S. aureus DsbA (SaDsbA), which incorporates a thioredoxin fold with an inserted helical domain, like its Escherichia coli counterpart EcDsbA, but it lacks the characteristic hydrophobic patch and has a truncated binding groove near the active site. These findings suggest that SaDsbA has a different substrate specificity than EcDsbA. Thermodynamic studies indicate that the oxidized and reduced forms of SaDsbA are energetically equivalent, in contrast to the energetically unstable disulfide form of EcDsbA. Further, the partial complementation of EcDsbA by SaDsbA is independent of EcDsbB and biochemical assays show that SaDsbA does not interact with EcDsbB. The identical stabilities of oxidized and reduced SaDsbA may facilitate direct re-oxidation of the protein by extracellular oxidants, without the need for DsbB.The formation of native disulfide bonds through air oxidation of cysteine pairs is a slow reaction and organisms ranging from bacteria to humans encode enzymatic systems to catalyze the process. In eukaryotes, oxidative folding in the endoplasmic reticulum is primarily catalyzed by protein-disulfide isomerases that are reoxidized by Ero1p/Erv2p proteins (reviewed in Ref. 1). In Escherichia coli, dithiol oxidation takes place in the periplasm through the action of the Dsb 3 (Disulfide bond) family of proteins. Dsb proteins form two distinct pathways, the DsbA-DsbB (oxidative) pathway that introduces disulfides indiscriminately, and the DsbC/DsbG-DsbD (isomerization) pathway that shuffles incorrect disulfides (2, 3).Probably the best-studied Dsb protein is EcDsbA, the primary disulfide catalyst in E. coli (reviewed in Refs. 2, 4). This promiscuously oxidizing protein is a 21-kDa monomer containing a CPHC active site in a thioredoxin (TRX) fold with an inserted helical domain of ϳ80 residues. Upon catalyzing disulfide bond formation in substrate proteins, reduced EcDsbA relies on EcDsbB, a quinone reductase, to recover its catalytically active, and higher energy, oxidized form (5

Section: Discussionmentioning

confidence: 99%

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Heras

Kurz

Jarrott

et al. 2008

“…Strain GB100 (As MC4100, pcnB1 zad-981::Tn10d (Km r ), dsbA::Cm, ⌬tatABC::aac (3)IV) was constructed from strain MC4100-P (28) as follows. Initially the dsbA::Cm allele from strain LM105 (29) was introduced by P1 transduction. Subsequently, a marked deletion of tatABC was also introduced by P1 transduction.…”

Section: Methodsmentioning

confidence: 99%

Cysteine-scanning Mutagenesis and Disulfide Mapping Studies of the Conserved Domain of the Twin-arginine Translocase TatB Component

Lee

Orriss

Buchanan

et al. 2006

The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane ␣-helix that precedes an amphipathic ␣-helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.

“…These enzymes catalyze thiol-disulfide oxidoreductase reactions that are important in enzyme catalysis and in the maintenance of the correct thiol redox state in proteins. Dsb (disulfide bond) proteins are a specific of TRX-like proteins that are essential for the oxidative folding of secreted proteins in many Gram-negative bacteria (1)(2)(3). In Escherichia coli K-12 five different Dsb proteins catalyze the correct introduction of disulfide bonds in substrate proteins (4).…”

Section: Thioredoxin (Trx)mentioning

confidence: 99%

Structural and Functional Characterization of Three DsbA Paralogues from Salmonella enterica Serovar Typhimurium

Heras

Totsika

Jarrott

et al. 2010

In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbAlike proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence.