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.     

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.     

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.     

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.     

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.     

Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy

The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13C and 15N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three-dimensional (3D) correlation experiments on a uniformly 13C, 15N-labeled sample at 750-MHz 1H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.     

NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation

We describe the NMR structure of DsbB, a polytopic helical membrane protein. DsbB, a bacterial cytoplasmic membrane protein, plays a key role in disulfide bond formation. It reoxidizes DsbA, the periplasmic protein disulfide oxidant, using the oxidizing power of membrane-embedded quinones. We determined the structure of an interloop disulfide bond form of DsbB, an intermediate in catalysis. Analysis of the structure and interactions with substrates DsbA and quinone reveals functionally relevant changes induced by these substrates. Analysis of the structure, dynamics measurements, and NMR chemical shifts around the interloop disulfide bond suggest how electron movement from DsbA to quinone through DsbB is regulated and facilitated. Our results demonstrate the extraordinary utility of NMR for functional characterization of polytopic integral membrane proteins and provide insights into the mechanism of DsbB catalysis.     

Structure and mechanisms of the DsbB-DsbA disulfide bond generation machine

All organisms possess specific cellular machinery that introduces disulfide bonds into proteins newly synthesized and transported out of the cytosol. In E. coli, the membrane-integrated DsbB protein cooperates with ubiquinone to generate a disulfide bond, which is transferred to DsbA, a periplasmic dithiol oxido-reductase that serves as the direct disulfide bond donor to proteins folding oxidatively in this compartment. Despite the extensive accumulation of knowledge on this oxidation system, molecular details of the DsbB reaction mechanisms had been controversial due partly to the lack of structural information until our recent determination of the crystal structure of a DsbA-DsbB-ubiquinone complex. In this review we discuss the structural and chemical nature of reaction intermediates in the DsbB catalysis and the illuminated molecular mechanisms that account for the de novo formation of a disulfide bond and its donation to DsbA. It is suggested that DsbB gains the ability to oxidize its specific substrate, DsbA, having very high redox potential, by undergoing a DsbA-induced rearrangement of cysteine residues. One of the DsbB cysteines that are now reduced then interacts with ubiquinone to form a charge transfer complex, leading to the regeneration of a disulfide at the DsbB active site, and the cycle can begin anew.     

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.     

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 disulfide bond formation (Dsb) system

In oxidative folding of proteins in the bacterial periplasmic space, disulfide bonds are introduced by the oxidation system and isomerized by the reduction system. These systems utilize the oxidizing and the reducing equivalents of quinone and NADPH, respectively, that are transmitted across the cytoplasmic membrane through integral membrane components DsbB and DsbD. In both pathways, alternating interactions between a Cys-XX-Cys-containing thioredoxin domain and other regulatory domain lead to the maintenance of oxidized and reduced states of the specific terminal enzymes, DsbA that oxidizes target cysteines and DsbC that reduces an incorrect disulfide to allow its isomerization into the physiological one. Molecular details of these remarkable biochemical cascades are being rapidly unraveled by genetic, biochemical, and structural analyses in recent years.     

Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity.

We identified and characterized an Erwinia chrysanthemi gene able to complement an Escherichia coli dsbA mutation that prevents disulfide bond formation in periplasmic proteins. This gene, dsbC, codes for a 24 kDa periplasmic protein that contains a characteristic active site sequence of disulfide isomerases, Phe-X-X-X-X-Cys-X-X-Cys. Besides the active site, DsbC has no homology with DsbA, thioredoxin or eukaryotic protein disulfide isomerase and it could define a new subfamily of disulfide isomerases. Purified DsbC protein is able to catalyse insulin oxidation in a dithiothreitol dependent manner. The E.coli gene xprA codes for a protein functionally equivalent to DsbC. The in vivo function of DsbC seems to be the formation of disulfide bonds in proteins. The presence of XprA could explain the residual disulfide isomerase activity existing in dsbA mutants. Re-oxidation of XprA does not seem to occur through DsbB, the protein that probably re-oxidizes DsbA.     

Identification of a segment of DsbB essential for its respiration-coupled oxidation

In the Escherichia coli protein disulphide bond formation pathway, membrane-bound DsbB oxidizes periplasmic DsbA, the disulphide bond-introducing enzyme. The Cys-41-Val-Leu-Cys-44 motif in the first periplasmic domain of DsbB is kept strongly oxidized by the respiratory function of the cell. We now show that the characteristic dithiothreitol resistance of the Cys-41-Cys-44 bond was retained even when the flanked Val-Leu combination was replaced by XX sequences from other oxidoreductases. Results of insertion mutagenesis showed that only the insertions (1-31 amino acids) in the region C-terminally adjacent to the CXXC motif impaired the oxidized state of DsbB. Deletion of a single amino acid from this region also rendered DsbB reduced and inactive. However, single amino acid substitutions of the four residues flanked by CXXC and the transmembrane segment did not abolish the oxidation of DsbB. These results suggest that some physical property, such as distance of the CXXC motif from the membrane, is important for the respiration-coupled oxidation of DsbB.     

Structure analysis of the extracellular domain reveals disulfide bond forming-protein properties of Mycobacterium tuberculosis Rv2969c

Disulfide bond-forming (Dsb) protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins. DsbA introduces disulfide bonds into folding proteins, and is re-oxidized through interaction with its redox partner DsbB. Mycobacterium tuberculosis, a Gram-positive bacterium, expresses a DsbA-like protein ( Rv2969c), an extracellular protein that has its Nterminus anchored in the cell membrane. Since Rv2969c is an essential gene, crucial for disulfide bond formation, research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection. In the present work, the crystal structures of theextracellular region of Rv2969c (Mtb DsbA) were determined in both its reduced and oxidized states. The overall structure of Mtb DsbA can be divided into two domains: a classical thioredoxin-like domain with a typical CXXC active site, and an α-helical domain. It largely resembles its Escherichiacoli homologue EcDsbA, however, it possesses a truncated binding groove; in addition, its active site is surrounded by an acidic, rather than hydrophobic surface. In our oxidoreductase activity assay, Mtb DsbA exhibited a different substrate specificity when compared to EcDsbA. Moreover, structural analysis revealed a second disulfide bond in Mtb DsbA, which is rare in the previously reported DsbA structures, and is assumed to contribute to the overall stability of Mtb DsbA. To investigate the disulphide formation pathway in M.tuberculosis, we modeled Mtb Vitamin K epoxide reductase (Mtb VKOR), a binding partner of Mtb DsbA, to Mtb DsbA.     

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.     

Folding of a bacterial outer membrane protein during passage through the periplasm.

The transport of bacterial outer membrane proteins to their destination might be either a one-step process via the contact zones between the inner and outer membrane or a two-step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane.     

The oxidase DsbA folds a protein with a nonconsecutive disulfide

One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at-165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb(-) strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed.     

Identification of a protein required for disulfide bond formation in vivo

We describe a mutation (dsbA) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled beta-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 Mr periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.