DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo

Oxidative protein folding in the periplasm of Escherichia coli is catalyzed by the thiol-disulfide oxidoreductases DsbA and DsbC. We investigated the catalytic efficiency of these enzymes during folding of proteins with a very complex disulfide pattern in vivo and in vitro, using the Ragi bifunctional inhibitor (RBI) as model substrate. RBI is a 13.1 kDa protein with five overlapping disulfide bonds. We show that reduced RBI can be refolded quantitatively in glutathione redox buffers in vitro and spontaneously adopts the single correct conformation out of 750 possible species with five disulfide bonds. Under oxidizing redox conditions, however, RBI folding is hampered by accumulation of a large number of intermediates with non-native disulfide bonds, while a surprisingly low number of intermediates accumulates under optimal or reducing redox conditions. DsbC catalyzes folding of RBI under all redox conditions in vitro, but is particularly efficient in rearranging buried, non-native disulfide bonds formed under oxidizing conditions. In contrast, the influence of DsbA on the refolding reaction is essentially restricted to reducing redox conditions where disulfide formation is rate limiting. The effects of DsbA and DsbC on folding of RBI in E.coli are very similar to those observed in vitro. Whereas overexpression of DsbA has no effect on the amount of correctly folded RBI, co-expression of DsbC enhanced the efficiency of RBI folding in the periplasm of E.coli about 14-fold. Addition of reduced glutathione to the growth medium together with DsbC overexpression further increased the folding yield of RBI in vivo to 26-fold. This shows that DsbC is the bacterial enzyme of choice for improving the periplasmic folding yields of proteins with very complex disulfide bond patterns.     

Periplasmic disulfide isomerase DsbC is involved in the reduction of copper binding protein CueP from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen with the ability to survive and replicate in macrophages. Periplasmic copper binding protein CueP is known to confer copper resistance to S. Typhimurium, and has been implicated in ROS scavenge activity by transferring the copper ion to a periplasmic superoxide dismutase or by directly reducing the copper ion. Structural and biochemical studies on CueP showed that its copper binding site is surrounded by conserved cysteine residues. Here, we present evidence that periplasmic disulfide isomerase DsbC plays a key role in maintaining CueP protein in the reduced state. We observed purified DsbC protein efficiently reduced the oxidized form of CueP, and that it acted on two (Cys104 and Cys172) of the three conserved cysteine residues. Furthermore, we found that a surface-exposed conserved phenylalanine residue in CueP was important for this process, which suggests that DsbC specifically recognizes the residue of CueP. An experiment using an Escherichia coli system confirmed the critical role played by DsbC in the ROS scavenge activity of CueP. Taken together, we propose a molecular insight into how CueP collaborates with the periplasmic disulfide reduction system in the pathogenesis of the bacteria.     

Conserved role of the linker alpha-helix of the bacterial disulfide isomerase DsbC in the avoidance of misoxidation by DsbB

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.     

Two periplasmic disulfide oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a component of the Salmonella pathogenicity island 2 type III secretion system

The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo.     

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

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.     

Folding mechanisms of periplasmic proteins

More than one fifth of the proteins encoded by the genome of Escherichia coli are destined to the bacterial cell envelope. Over the past 20 years, the mechanisms by which envelope proteins reach their three-dimensional structure have been intensively studied, leading to the discovery of an intricate network of periplasmic folding helpers whose members have distinct but complementary roles. For instance, the correct assembly of ß-barrel proteins containing disulfide bonds depends both on chaperones like SurA and Skp for transport across the periplasm and on protein folding catalysts like DsbA and DsbC for disulfide bond formation. In this review, we provide an overview of the current knowledge about the complex network of protein folding helpers present in the periplasm of E. coli and highlight the questions that remain unsolved. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.     

Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.     

In vivo oxidative protein folding can be facilitated by oxidation–reduction cycling

Current dogma dictates that bacterial proteins with misoxidized disulfide bonds are shuffled into correctly oxidized states by DsbC. There are two proposed mechanisms for DsbC activity. The first involves a DsbC-only model of substrate disulfide rearrangement. The second invokes cycles of reduction and oxidation of substrate disulfide bonds by DsbC and DsbA respectively. Here, we addressed whether the second mechanism is important in vivo by identifying whether a periplasmic reductase could complement DsbC. We screened for naturally occurring periplasmic reductases in Bacteroides fragilis , a bacterium chosen because we predicted it encodes reductases and has a reducing periplasm. We found that the B. fragilis periplasmic protein TrxP has a thioredoxin fold with an extended N-terminal region; that it is a very active reductase but a poor isomerase; and that it fully complements dsbC . These results provide direct in vivo evidence that correctly folded protein is achievable via cycles of oxidation and reduction.     

Oxidative refolding of lysozyme assisted by DsbA, DsbC and the GroEL apical domain immobilized in cellulose

Expression of recombinant proteins in Escherichia coli often leads to formation of inclusion bodies (IB). If a recombinant protein contains one or more disulfide bonds, protein refolding and thiol oxidation reactions are required to recover its biological activity. Previous studies have demonstrated that molecular chaperones and foldases assist with the in vitro protein refolding. However, their use has been limited by the stoichiometric amount required for the refolding reaction. In search of alternatives to facilitate the use of these folding biocatalysts in this study, DsbA, DsbC, and the apical domain of GroEL (AD) were fused to the carbohydrate-binding module CBD_Cex of Cellulomonas fimi. The recombinant proteins were purified and immobilized in cellulose and used to assist the oxidative refolding of denatured and reduced lysozyme. The assisted refolding yields obtained with immobilized folding biocatalysts were at least twice of those obtained in the spontaneous refolding, suggesting that the AD, DsbA, and DsbC immobilized in cellulose might be useful for the oxidative refolding of recombinant proteins that are expressed as inclusion bodies. In addition, the spontaneous or assisted refolding kinetics data fitted well (r² > 0.9) to a previously reported lysozyme refolding model. The estimated refolding (k _N) and aggregation (k _A) constants were consistent with the hypothesis that foldases assisted the oxidative refolding of lysozyme by decreasing protein aggregation rather than increasing the refolding rate.     

大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用

大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。     

The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase, DsbC

The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.     

In vivo substrate specificity of periplasmic disulfide oxidoreductases

In Escherichia coli, a family of periplasmic disulfide oxidoreductases catalyzes correct disulfide bond formation in periplasmic and secreted proteins. Despite the importance of native disulfide bonds in the folding and function of many proteins, a systematic investigation of the in vivo substrates of E. coli periplasmic disulfide oxidoreductases, including the well characterized oxidase DsbA, has not yet been performed. We combined a modified osmotic shock periplasmic extract and two-dimensional gel electrophoresis to identify substrates of the periplasmic oxidoreductases DsbA, DsbC, and DsbG. We found 10 cysteine-containing periplasmic proteins that are substrates of the disulfide oxidase DsbA, including PhoA and FlgI, previously established DsbA substrates. This technique did not detect any in vivo substrates of DsbG, but did identify two substrates of DsbC, RNase I and MepA. We confirmed that RNase I is a substrate of DsbC both in vivo and in vitro. This is the first time that DsbC has been shown to affect the in vivo function of a native E. coli protein, and the results strongly suggest that DsbC acts as a disulfide isomerase in vivo. We also demonstrate that DsbC, but not DsbG, is critical for the in vivo activity of RNase I, indicating that DsbC and DsbG do not function identically in vivo. The absence of substrates for DsbG suggests either that the in vivo substrate specificity of DsbG is more limited than that of DsbC or that DsbG is not active under the growth conditions tested. Our work represents one of the first times the in vivo substrate specificity of a folding catalyst system has been systematically investigated. Because our methodology is based on the simple assumption that the absence of a folding catalyst should cause its substrates to be present at decreased steady-state levels, this technique should be useful in analyzing the substrate specificity of any folding catalyst or chaperone for which mutations are available.     

Monitoring the Disulfide Bond Formation of a Cysteine-Rich Repeat Protein from Helicobacter pylori in the Periplasm of Escherichia coli

Helicobacter pylori infection increases the risk of cardiovascular diseases besides leading to duodenal and gastric peptic ulcerations. H. pylori cysteine-rich protein B (HcpB) is a disulfide-rich repeat protein that belongs to the family of Sel1-like repeat proteins. HcpB contains four pairs of anti-parallel alpha helices that fold into four repeats with disulfide bonds bridging the helices of each repeat. Recent in vitro oxidative refolding of HcpB identified that the formation and folding of the disulfide bond in the N-terminal repeat are the rate limiting step. Here we attempted to understand the disulfide formation of HcpB in the periplasm of Escherichia coli. The protein was expressed in wild type (possessed enzymes DsbA, B, C, and D) and knock out (Dsb enzymes deleted one at a time) E. coli strains. The soluble part of the periplasm when analyzed by SDS-PAGE and Western Blot showed that the wild type and DsbC/D knock out strains contained native oxidized HcpB while the protein was absent in the DsbA/B knock out strains. Hence the recombinant expression of HcpB in E. coli requires DsbA and DsbB for disulfide bond formation and it is independent of DsbC and DsbD. Prolonged cell growth resulted in the proteolytic degradation of the N-terminal repeat of HcpB. The delayed folding of the N-terminal repeat observed during in vitro oxidative refolding could be the reason for the enhanced susceptibility to proteolytic cleavage in the periplasm. In summary, a good correlation between in vivo and in vitro disulfide bond formation of HcpB is observed.     

The Multidrug Resistance IncA/C Transferable Plasmid Encodes a Novel Domain-swapped Dimeric Protein-disulfide Isomerase

The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (−161 mV) is more reducing than EcDsbC (−130 mV) and EcDsbG (−126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.     

Staphylococcus aureus DsbA does not have a destabilizing disulfide. A new paradigm for bacterial oxidative folding

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.     

Mutants in DsbB that appear to redirect oxidation through the disulfide isomerization pathway

Disulfide bond formation occurs in secreted proteins in Escherichia coli when the disulfide oxidoreductase DsbA, a soluble periplasmic protein, nonspecifically transfers a disulfide to a substrate protein. The catalytic disulfide of DsbA is regenerated by the inner-membrane protein DsbB. To help identify the specificity determinants in DsbB and to understand the nature of the kinetic barrier preventing direct oxidation of newly secreted proteins by DsbB, we imposed selective pressure to find novel mutations in DsbB that would function to bypass the need for the disulfide carrier DsbA. We found a series of mutations localized to a short horizontal α-helix anchored near the outer surface of the inner membrane of DsbB that eliminated the need for DsbA. These mutations changed hydrophobic residues into nonhydrophobic residues. We hypothesize that these mutations may act by decreasing the affinity of this α-helix to the membrane. The DsbB mutants were dependent on the disulfide oxidoreductase DsbC, a soluble periplasmic thiol-disulfide isomerase, for complementation. DsbB is not normally able to oxidize DsbC, possibly due to a steric clash that occurs between DsbC and the membrane adjacent to DsbB. DsbC must be in the reduced form to function as an isomerase. In contrast, DsbA must remain oxidized to function as an oxidizing thiol-disulfide oxidoreductase. The lack of interaction that normally exists between DsbB and DsbC appears to provide a means to separate the DsbA-DsbB oxidation pathway and the DsbC-DsbD isomerization pathway. Our mutants in DsbB may act by redirecting oxidant flow to take place through the isomerization pathway.     

The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.     

Thiol-disulfide exchange in an immunoglobulin-like fold: structure of the N-terminal domain of DsbD

Escherichia coli DsbD transports electrons across the plasma membrane, a pathway that leads to the reduction of protein disulfide bonds. Three secreted thioredoxin-like factors, DsbC, DsbE, and DsbG, reduce protein disulfide bonds whereby an active site C-X-X-C motif is oxidized to generate a disulfide bond. DsbD catalyzes the reduction of the disulfide of DsbC, DsbE, and DsbG but not of the thioredoxin-like oxidant DsbA. The reduction of DsbC, DsbE, and DsbG occurs by transport of electrons from cytoplasmic thioredoxin to the C-terminal thioredoxin-like domain of DsbD (DsbD(C)). The N-terminal domain of DsbD, DsbD(N), acts as a versatile adaptor in electron transport and is capable of forming disulfides with oxidized DsbC, DsbE, or DsbG as well as with reduced DsbD(C). Isolated DsbD(N) is functional in electron transport in vitro. Crystallized DsbD(N) assumes an immunoglobulin-like fold that encompasses two active site cysteines, C103 and C109, forming a disulfide bond between beta-strands. The disulfide of DsbD(N) is shielded from the environment and capped by a phenylalanine (F70). A model is discussed whereby the immunoglobulin fold of DsbD(N) may provide for the discriminating interaction with thioredoxin-like factors, thereby triggering movement of the phenylalanine cap followed by disulfide rearrangement.     

Biochemical and Structural Study of the Homologues of the Thiol-Disulfide Oxidoreductase DsbA in Neisseria meningitidis

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. The Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host interactive biology, while the function of DsbA3 remains unknown.This work reports the biochemical characterization of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. As predicted by sequence homology, both enzymes adopt the classic Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of − 80 mV, the neisserial DsbAs are the most oxidizing thioredoxin-like enzymes known to date. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. For each of these enzymes, this study shows that a threonine residue found within the active-site region plays a key role in dictating this extraordinary oxidizing power. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family.