Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.     

Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

Paradoxical redox properties of DsbB and DsbA in the protein disulfide-introducing reaction cascade

Protein disulfide bond formation in the bacterial periplasm is catalyzed by the Dsb enzymes in conjunction with the respiratory quinone components. Here we characterized redox properties of the redox active sites in DsbB to gain further insights into the catalytic mechanisms of DsbA oxidation. The standard redox potential of DsbB was determined to be -0.21 V for Cys41/Cys44 in the N-terminal periplasmic region (P1) and -0.25 V for Cys104/Cys130 in the C-terminal periplasmic region (P2), while that of Cys30/Cys33 in DsbA was -0.12 V. To our surprise, DsbB, an oxidant for DsbA, is intrinsically more reducing than DsbA. Ubiquinone anomalously affected the apparent redox property of the P1 domain, and mutational alterations of the P1 region significantly lowered the catalytic turnover. It is inferred that ubiquinone, a high redox potential compound, drives the electron flow by interacting with the P1 region with the Cys41/Cys44 active site. Thus, DsbB can mediate electron flow from DsbA to ubiquinone irrespective of the intrinsic redox potential of the Cys residues involved.     

Mechanism of the electron transfer catalyst DsbB from Escherichia coli

The membrane protein DsbB from Escherichia coli is essential for disulfide bond formation and catalyses the oxidation of the periplasmic dithiol oxidase DsbA by ubiquinone. DsbB contains two catalytic disulfide bonds, Cys41-Cys44 and Cys104-Cys130. We show that DsbB directly oxidizes one molar equivalent of DsbA in the absence of ubiquinone via disulfide exchange with the 104-130 disulfide bond, with a rate constant of 2.7 x 10 M(-1) x s(-1). This reaction occurs although the 104-130 disulfide is less oxidizing than the catalytic disulfide bond of DsbA (E(o)' = -186 and -122 mV, respectively). This is because the 41-44 disulfide, which is only accessible to ubiquinone but not to DsbA, is the most oxidizing disulfide bond in a protein described so far, with a redox potential of -69 mV. Rapid intramolecular disulfide exchange in partially reduced DsbB converts the enzyme into a state in which Cys41 and Cys44 are reduced and thus accessible for reoxidation by ubiquinone. This demonstrates that the high catalytic efficiency of DsbB results from the extreme intrinsic oxidative force of the enzyme.     

DsbB catalyzes disulfide bond formation de novo

DsbA and DsbB are responsible for disulfide bond formation. DsbA is the direct donor of disulfides, and DsbB oxidizes DsbA. DsbB has the unique ability to generate disulfides by quinone reduction. It is thought that DsbB oxidizes DsbA via thiol disulfide exchange. In this mechanism, a disulfide is formed across the N-terminal pair of cysteines (Cys-41/Cys-44) in DsbB by quinone reduction. This disulfide is then transferred on to the second pair of cysteine residues in DsbB (Cys-104/Cys-130) and then finally transferred to DsbA. We have shown here the redox potential of the two disulfides in DsbB are -271 and -284 mV, respectively, and considerably less oxidizing than the disulfide of DsbA at -120 mV. In addition, we have found the Cys-104/Cys-130 disulfide of DsbB to actually be a substrate for DsbA in vitro. These findings indicate that the disulfides in DsbB are unsuitable to function as the oxidant of DsbA. Furthermore, we have shown that mutants in DsbB that lack either pair or all of its cysteines are also capable of oxidizing DsbA. These unexpected findings raise the possibility that the oxidation of DsbA by DsbB does not occur via thiol disulfide exchange as is widely assumed but rather, directly via quinone reduction.     

Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.     

Crucial effect of the first CXXC motif of human QSOX 1b on the activity to different substrates

Among all sulphhydryl oxidases involved in disulphide formation, quiescin-sulphhydryl oxidase (QSOX) is unique for its multidomain structure, protein thiol oxidation activity and highly efficient catalysis. In this study, site-directed mutagenesis and molecular modelling methods were integrated to investigate the structural and functional characteristics of QSOX, especially the importance of the three CXXC motifs. Site-directed mutagenesis suggested that the C449-C452 motif was essential for the activity of human QSOX 1b; the C70-C73 motif was fundamental in electron transfer from thiol-containing substrate including reduced proteins, DTT, GSH rather than the phosphine-based thiol reductant TCEP, to the C449-C452 motif; and the C509-C512 motif was not involved in electron transfer during disulphide formation. The different roles of the CXXC motifs indicated that there were discrepant electron transfer pathways for the oxidation of thiol-containing substrates and non-thiol disulphide reductants. Molecular modelling method was then used to draw a reasonable picture for the electron transfer process and to elucidate the mechanism of electron transfer when different substrates were oxidized, which will greatly enhance our understanding of the action mechanism of QSOX.     

Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation

Oxidation of cysteine pairs to disulfide requires cellular factors present in the bacterial periplasmic space. DsbB is an E. coli membrane protein that oxidizes DsbA, a periplasmic dithiol oxidase. To gain insight into disulfide bond formation, we determined the crystal structure of the DsbB-DsbA complex at 3.7 A resolution. The structure of DsbB revealed four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. We propose that DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.     

Characterization of the menaquinone-dependent disulfide bond formation pathway of Escherichia coli

In the protein disulfide-introducing system of Escherichia coli, plasma membrane-integrated DsbB oxidizes periplasmic DsbA, the primary disulfide donor. Whereas the DsbA-DsbB system utilizes the oxidizing power of ubiquinone (UQ) under aerobic conditions, menaquinone (MK) is believed to function as an immediate electron acceptor under anaerobic conditions. Here, we characterized MK reactivities with DsbB. In the absence of UQ, DsbB was complexed with MK8 in the cell. In vitro studies showed that, by binding to DsbB in a manner competitive with UQ, MK specifically oxidized Cys41 and Cys44 of DsbB and activated its catalytic function to oxidize reduced DsbA. In contrast, menadione used in earlier studies proved to be a more nonspecific oxidant of DsbB. During catalysis, MK8 underwent a spectroscopic transition to develop a visible violet color (lambdamax = 550 nm), which required a reduced state of Cys44 as shown previously for UQ color development (lambdamax = 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30 degrees C, two reaction components were observed, one completing within minutes and the other taking >1 h. Both of these reaction modes were accompanied by the transition state of MK, for which the slower reaction proceeded through the disulfide-linked DsbA-DsbB(MK) intermediate. The MK-dependent pathway provides opportunities to further dissect the quinone-dependent DsbA-DsbB redox reactions.     

Bacterial conjugation: a two-step mechanism for DNA transport

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing. Two isomerases exist in Escherichia coli, DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.     

Four cysteines of the membrane protein DsbB act in concert to oxidize its substrate DsbA

Protein disulfide bond formation in Escherichia coli is catalyzed by the periplasmic protein DsbA. A cytoplasmic membrane protein DsbB maintains DsbA in the oxidized state by transferring electrons from DsbA to quinones in the respiratory chain. Here we show that DsbB activity can be reconstituted by co-expression of N- and C-terminal fragments of the protein, each containing one of its redox-active disulfide bonds. This system has allowed us (i) to demonstrate that the two DsbB redox centers interact directly through a disulfide bond formed between the two DsbB domains and (ii) to identify the specific cysteine residues involved in this covalent interaction. Moreover, we are able to capture an intermediate in the process of electron transfer from one redox center to the other. These results lead us to propose a model that describes how the cysteines cooperate in the early stages of oxidation of DsbA. DsbB appears to adopt a novel mechanism to oxidize DsbA, using its two pairs of cysteines in a coordinated reaction to accept electrons from the active cysteines in DsbA.     

Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies.

The single interchain disulphide bond in platelet glycoprotein IIb (GPIIb) is accessible to extracellular reductants, and selective cleavage does not liberate GPIIb alpha from platelet plasma membrane, confirming that non-covalent interactions contribute to maintaining attachment of this subunit to the membrane. Eosin-maleimide labelling of isolated GPIIb after selective cleavage of this interchain disulphide bond, followed by full reduction and alkylation, CNBr cleavage, and analysis of the cleavage products allowed us to establish that this interchain disulphide bridge is formed between GPIIb beta (GPIIb beta-subunit) Cys-9 and GPIIb alpha Cys-826, and this conclusion was confirmed by independent routes. The other two cysteines of GPIIb beta (Cys-14 and Cys-19) form the single intrachain disulphide bond in this subunit. Last, the intrachain disulphides in GPIIb alpha (GPIIb alpha-subunit) are distributed in four main peptide domains which are not disulphide-bonded among themselves. The linear epitope for monoclonal antibody M1 is localized between Pro-4 and Met-24 (or Met-31) of GPIIb beta. The linear epitope for M3 is situated between Cys-826 and the C-terminus of GPIIb alpha. The M4 epitope is also linear and localized somewhere between residues 115 and 285 of GPIIb alpha. Finally, the epitopes for M5 and M6 are somewhere between Cys-608 and Met-704, within a 35 kDa membrane-bound chymotryptic product of digestion of GPIIb in whole platelets. The N-terminal amino acid sequences determined for eight different cleavage products of GPIIb alpha and GPIIb beta agree with the corresponding amino acid sequences predicted by cDNA sequence for human-erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482].     

Dynamic nature of disulphide bond formation catalysts revealed by crystal structures of DsbB

In the Escherichia coli system catalysing oxidative protein folding, disulphide bonds are generated by the cooperation of DsbB and ubiquinone and transferred to substrate proteins through DsbA. The structures solved so far for different forms of DsbB lack the Cys104–Cys130 initial‐state disulphide that is directly donated to DsbA. Here, we report the 3.4 Å crystal structure of a DsbB–Fab complex, in which DsbB has this principal disulphide. Its comparison with the updated structure of the DsbB–DsbA complex as well as with the recently reported NMR structure of a DsbB variant having the rearranged Cys41–Cys130 disulphide illuminated conformational transitions of DsbB induced by the binding and release of DsbA. Mutational studies revealed that the membrane‐parallel short α‐helix of DsbB has a key function in physiological electron flow, presumably by controlling the positioning of the Cys130‐containing loop. These findings demonstrate that DsbB has developed the elaborate conformational dynamism to oxidize DsbA for continuous protein disulphide bond formation in the cell.     

Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi

Secretion to the cell exterior of cellulase EGZ and of at least six pectinases enables the Gram-negative Erwinia chrysanthemi to cause severe plant disease. The C-terminal cellulose-binding domain (CBD) of EGZ was found to contain a disulphide bond which forms, in the periplasm, between residues Cys-325 and Cys-382. Dithiothreitol (DTT)-treatment of native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultures led to immediate arrest of secretion of EGZ which accumulated in the periplasm where the CBD was eventually proteolysed. Site-directed mutagenesis that affected Cys residues involved in disulphide bond formation resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted, Instead they accumulated in the periplasm. Interestingly, the region around EGZ Cys-325 is conserved in two pectinases secreted by the same pathway as EGZ. We conclude that the conserved Cys, and possibly adjacent residues, bear essential information for EGZ to be secreted and that periplasmic disulphide bond formation is an obligatory step which provides a pre-folded functional form of EGZ with secretion competence.     

Laboratory evolution of Escherichia coli thioredoxin for enhanced catalysis of protein oxidation in the periplasm reveals a phylogenetically conserved substrate specificity determinant

Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA(-) cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA(-) cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.     

Active-site properties of the oxidized and reduced C-terminal domain of DsbD obtained by NMR spectroscopy

The periplasmic C-terminal domain of the Escherichia coli DsbD protein (cDsbD) has a thioredoxin fold. The two cysteine residues in the CXXC motif serve as the reductant for the disulfide bond of the N-terminal domain which can in turn act as a reductant for various periplasmic partners. The resulting disulfide bond in cDsbD is reduced via an unknown mechanism by the transmembrane helical domain of the protein. We show by NMR analysis of (13)C, (15)N-labelled cDsbD that the protein is rigid, is stable to extremes of pH and undergoes only localized conformational changes in the vicinity of the CXXC motif, and in adjacent regions of secondary structure, upon undergoing the reduced/oxidized transition. pK(a) values have been determined, using 2D NMR, for the N-terminal cysteine of the CXXC motif, Cys461, as well as for other active-site residues. It is demonstrated using site-directed mutagenesis that the negative charges of the side-chains of Asp455 and Glu468 in the active site contribute to the unusually high pK(a) value, 10.5, of Cys461. This value is higher than expected from knowledge of the reduction potential of cDsbD. In a double mutant of cDsbD, D455N/E468Q, the pK(a) value of Cys461 is lowered to 8.6, a value close to that expected for an unperturbed cysteine residue. The pK(a) value of the second cysteine in wild-type cDsbD, Cys464, is significantly higher than the maximum pH value that was studied (pH 12.2).     

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.     

Preparation and structure of the charge-transfer intermediate of the transmembrane redox catalyst DsbB

Disulfide bond formation is a critical step in the folding of many secretory proteins. In bacteria, disulfide bonds are introduced by the periplasmic dithiol oxidase DsbA, which transfers its catalytic disulfide bond to folding polypeptides. Reduced DsbA is reoxidized by ubiquinone Q8, catalyzed by inner membrane quinone reductase DsbB. Here, we report the preparation of a kinetically stable ternary complex between wild-type DsbB, containing all essential cysteines, Q8 and DsbA covalently bound to DsbB. The crystal structure of this trapped DsbB reaction intermediate exhibits a charge-transfer interaction between Q8 and the Cys44 in the DsbB reaction center providing experimental evidence for the mechanism of de novo disulfide bond generation in DsbB.