DsbD-catalyzed transport of electrons across the membrane of Escherichia coli.

Dsb proteins catalyze folding and oxidation of polypeptides in the periplasm of Escherichia coli. DsbC reduces wrongly paired disulfides by transferring electrons from its catalytic dithiol motif (98)CGYC. Genetic evidence suggests that recycling of this motif requires at least three proteins, the cytoplasmic thioredoxin reductase (TrxB) and thioredoxin (TrxA) as well as the DsbD membrane protein. We demonstrate here that electrons are transferred directly from thioredoxin to DsbD and from DsbD to DsbC. Three cysteine pairs within DsbD undergo reversible disulfide rearrangements. Our results suggest a novel mechanism for electron transport across membranes whereby electrons are transferred sequentially from cysteine pairs arranged in a thioredoxin-like motif (CXXC) to a cognate reactive disulfide.

Dsb proteins catalyze folding and oxidation of polypeptides in the periplasm of Escherichia coli. DsbC reduces wrongly paired disulfides by transferring electrons from its catalytic dithiol motif 98 CGYC. Genetic evidence suggests that recycling of this motif requires at least three proteins, the cytoplasmic thioredoxin reductase (TrxB) and thioredoxin (TrxA) as well as the DsbD membrane protein. We demonstrate here that electrons are transferred directly from thioredoxin to DsbD and from DsbD to DsbC. Three cysteine pairs within DsbD undergo reversible disulfide rearrangements. Our results suggest a novel mechanism for electron transport across membranes whereby electrons are transferred sequentially from cysteine pairs arranged in a thioredoxin-like motif (CXXC) to a cognate reactive disulfide.
Disulfide bonds are important features in the folded structure of secreted polypeptides but are generally absent from proteins residing in the cytoplasm (1). Proteins acquire disulfide bonds after entry into the endoplasmic reticulum of eukaryotic cells (2,3). In bacterial cells, disulfide bond formation occurs in the periplasm, a compartment that is located between the cytoplasmic and outer membranes of Escherichia coli (4,5). Disulfide bond formation requires oxidation of a pair of cysteine sulfhydryl residues, a reaction catalyzed by bacterial DsbA (6,7). Electrons generated by oxidative folding are transferred from DsbA to DsbB, an inner membrane protein (8,9) containing four essential cysteine residues forming two reversible disulfide bonds (10,11). These disulfide bonds become reduced upon reoxidation of DsbA. DsbB uses quinones or menaquinones, hydrophobic compounds embedded within the cytoplasmic membrane, as electron acceptors for its recycling (12,13). Hence, DsbB functions to couple the DsbA-dependent oxidation of protein thiols in the periplasm to the electron transport chain in the cytoplasmic membrane.
DsbA displays no proofreading activity for the repair of wrongly paired disulfides (14). However, this as well as other reactions are catalyzed by DsbC (14,15), DsbE (CcmG) (16), and DsbG (17,18). For example, reduced DsbC (DsbC-(SH) 2 ) donates electrons to misfolded polypeptides resulting in the transfer of disulfides from misfolded polypeptides to DsbC (14). During this rearrangement, the active site 98 CGYC 101 thiol (sulfhydryl) residues of DsbC are oxidized (DsbC-S 2 ). What is the origin of electrons that are required for the proofreading activity of DsbC? Regeneration of reduced DsbC requires the DsbD membrane protein (19) as well as cytoplasmic thioredoxin (TrxA) 1 and thioredoxin reductase (TrxB) (20). TrxB transfers electrons from the NADPH coenzyme to oxidized thioredoxin (TrxA-S 2 ) thereby contributing to the reducing environment of the cellular cytoplasm (21,22). This study examines the specific mechanism whereby DsbD mediates electron transfer from the cytoplasm across the membrane.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth Conditions-Strains and plasmids used are listed in Table I. Sequences of primers used in this study can be obtained from the authors upon request. Cells were grown in Luria-Bertani (LB) medium (23) at 30°C using the appropriate antibiotic at the following concentrations: ampicillin, 100 g/ml; spectinomycin, 50 g/ml; chloramphenicol, 20 g/ml; and kanamycin, 50 g/ml. Induction of His 6 -tagged DsbD using the pSE420 plasmid (Invitrogen) was accomplished by addition of isopropyl-1-thio-␤-D-galactopyranoside to a final concentration of 1 mM. Plasmids expressing TrxA C32S (pTrxA C32S ) and TrxA C35S (pTrxA C35S ) were obtained from M. Russel (Rockefeller University). Plasmids pRK35 and pCC68 expressing wild-type DsbC and DsbC C101A were derivatives of plasmids pDM801 and pDM1461 described earlier (14,15). When needed, mutations were transduced into various backgrounds using P1 bacteriophage as described (23).
Replacement of DsbD Cysteines with Alanine-Plasmids encoding DsbD variants with two cysteine residues changed to alanine were generated by site-directed mutagenesis using the method described by Ansaldi et al. (24). Plasmid pDM2200 was used as a template (25). This plasmid is a derivative of pWSK30 (pSC101 replicon) (26) and carries a 3.1-kilobase pair DNA fragment containing the entire coding region for dsbD with upstream sequences. All plasmids were verified by sequencing. For purification of mutant DsbD proteins, clones encoding the full-length dsbD gene (wild-type or mutant) with a C-terminal His 6 tag, His 6 -tagged DsbD, were generated by polymerase chain reaction and cloned into pSE420 (Invitrogen) using EcoRI and BamHI restriction sites as described earlier (25).
Modification of Sulfhydryl Groups-Sulfhydryl groups were modified using either 2-aminoethyl methanethiosulfonate hydrobromide (MT-SEA) or 4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonic acid (AMS) reagents. Cells were grown to A 595 nm between 0.5 and 1.5 and incubated in the presence of 2.5 mM MTSEA (freshly prepared in water) for 5 min at room temperature. For analytical purposes, samples were precipitated with 7.5% trichloroacetic acid. Precipitates were collected by centrifugation, washed with acetone, spun, and suspended in 0.5 M Tris-HCl buffer, pH 7.0, containing 4% SDS. Samples were analyzed by 8 or 12% SDS-PAGE as well as by Western blot. For purification of the mixed disulfide species, cells were harvested at 4°C directly after MTSEA treatment. Such pellets were washed and suspended in buffer A (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% glycerol). Cells were kept frozen at Ϫ80°C until further use. The redox state of DsbD in vivo was assessed using AMS as described previously (12). * This work was supported by United States Public Health Service Grant GM58266 (to D. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Protein Purification-Wild-type DsbD (pJC350), DsbD C103A (pCC61), and DsbD C285A (pCC15) with an appended C-terminal His 6 tag were expressed by cloning the structural genes into pSE420 (Invitrogen). Cells expressing either one of the various His 6 -tagged DsbD proteins were grown to A 595 nm and incubated for 2 h with 1 mM isopropyl-1-thio-␤-D-galactopyranoside. Prior to lysis by French pressure (p.s.i. 14,000), cells were incubated with 2.5 mM MTSEA for 5 min at room temperature. Cleared cell extracts were centrifuged at 100,000 ϫ g for 45 min at 4°C. Membrane proteins in the sediments were suspended in 50 mM Tris-HCl, 150 mM NaCl, 2% octylglucoside, and 10 mM imidazole, pH 7.5. Insoluble material was removed by centrifugation prior to affinity purification of the various His 6 -tagged DsbD proteins on nickel-nitrilotriacetic acid.

RESULTS
Experimental Strategy-Genetic analyses suggest that to maintain DsbC, DsbG, and DsbE in a reduced state, electrons must flow from cytoplasmic NADPH to the periplasm (18,25,27). This process is dependent on TrxB, TrxA, and DsbD. TrxB is a cytoplasmic, dimeric protein. Each monomer includes one redox active disulfide ( 135 CATC 138 ) and one FAD molecule and catalyzes the reduction of TrxA by NADPH (22,28). TrxA is a small cytoplasmic protein (ϳ10 kDa) that contains one redox active site 32 CGPC 35 (21). Electrons flow from NADPH to TrxA by a mechanism involving the formation of a mixed disulfide intermediate between the NADH-activated TrxB enzyme and its substrate, TrxA. The TrxB thiolate Cys 135 attacks the disulfide of TrxA-S 2 to form transiently the mixed disulfide between TrxB and TrxA, Cys 135 -Cys 32 . This mixed intermediate is resolved by the Cys 138 thiolate, resulting in the release of reduced TrxA-(SH 2 ) (22,29).
Mixed disulfide intermediates are tethered by a disulfide bond to the active site dithiol of redox active proteins. Because such intermediates are extremely short lived, they are never observed in vivo. One way to capture these reaction intermediates is to make use of redox proteins containing only half of the redox active site. For example, substituting Cys 138 of TrxB and Cys 35 of TrxA for serine captures the mixed disulfide between TrxB and TrxA, the C 135 -C 32 intermediate (29). Similarly, substitution of the second cysteine (Cys 33 ) of the dithiol of DsbA with alanine ( 30 CPHA) leads to the production of a stable complex with a disulfide bond between Cys 30 of DsbA and Cys 104 of DsbB (10,11). This intermediate cannot be resolved due to the absence of the second thiol of DsbA. In contrast, mutation of the second cysteine in the dithiol motif of either DsbC (DsbC C101A ) or TrxA (TrxA C35S ) results in a large spectrum of intermolecular disulfide species as the reduced dithiol of these reductants recognizes many different polypeptide substrates (see Electron Transfer between TrxA and DsbD).
To elucidate the mechanism of electron transfer to periplasmic DsbC, we sought to capture reaction intermediates as an intermolecular disulfide between DsbD and DsbC.
A Stable Mixed Disulfide between DsbD and DsbC-The overall topology of DsbD has been previously shown by constructing fusions of DsbD membrane-spanning segments to the mature part of alkaline phosphatase (25,30,31). Two of these studies have revealed that both the N-and C-terminal domains of DsbD (each ϳ10 kDa in size) are positioned in the periplasm (Fig. 1A), suggesting that DsbD is initiated in the secretory pathway via a cleavable signal sequence prior its insertion in the cytoplasmic membrane (25,31). Both N-and C-terminal domains harbor two pairs of cysteines, Cys 103 -Cys 109 and Cys 461 -Cys 464 , respectively (Fig. 1A). The two domains are connected by eight transmembrane (TM) segments containing three cysteine residues, Cys 163 (TM1), Cys 282 (TM4), and Cys 285 (TM4) (Fig. 1A). DsbD residue Cys 282 (C4) is dispensable for electron transfer. In contrast, alanine substitution of cysteine residues at any one of the other cysteines (C 103 (C1), Cys 109 (C2), Cys 163 (C3), Cys 285 (C5), Cys 461 (C6), and Cys 464 (C7)) abolishes DsbD-mediated electron transfer, causing accumulation of DsbC-S 2 (25,30). When examined by immunoblotting of cellular extracts, none of the DsbD substitution mutants formed a stable intermolecular intermediate with DsbC (Fig.  1B). Thus, the presumed disulfide intermediate between DsbD and DsbC appears to be unstable and may be resolved rapidly by the attack of the second thiol, Cys 101 of DsbC.
To test this possibility, E. coli strains expressing DsbC C101A and various DsbD cysteine substitution mutants were examined. An intermolecular intermediate was observed in cells expressing DsbC C101A and DsbD C103A (Fig. 1C) whereas alanine substitution at any other cysteine of DsbD did not produce a mixed disulfide (Fig. 1B). To characterize the nature of the  (Fig. 2). Immunoblotting of eluted fractions revealed the presence of two electrophoretic species with an apparent molecular mass of 56 (DsbD C103A , alone) and 80 kDa (DsbD C103A -DsbC C101A ), respectively. Antibodies raised against DsbD (␣-DsbD) and DsbC (␣-DsbC) recognized the 80-kDa DsbD C103A -DsbC C101A species. In contrast, only ␣-DsbD bound to 56-kDa DsbD C103A . Incubation with the reducing agent dithiothreitol (DTT) resolved the 80-kDa mixed disulfide and resulted in the appearance of a 24-kDa polypeptide (Fig. 2). ␣-DsbC, but not ␣-DsbD, bound to the 24-kDa protein, indicating that this species represents DsbC C101A released from its disulfide linkage with DsbD C103A . Cysteine 98 of DsbC, the first sulfhydryl within the dithiol motif ( 98 CGYC), must be one Electron Transfer between TrxA and DsbD-E. coli lacking thioredoxin (⌬trxA) accumulate oxidized DsbC (20,25) and DsbD (Fig. 3A). Both cysteines of the dithiol motif of thioredoxin ( 32 CGPC 35 ) are required for electron transfer, as either mutant TrxA C32S or mutant TrxA C35S accumulated oxidized DsbD (Fig. 3A). Substitution of TrxA C35S but not TrxA C32S produced a spectrum of disulfide-linked intermolecular intermediates (Fig. 3B). Thus, substitution of the second cysteine of thioredoxin with serine (TrxA C35S ) results in a mutant protein capable of attacking numerous cytoplasmic disulfides but unable to resolve the mixed disulfide intermediates. Although cells expressing TrxA C35S accumulated DsbD in an oxidized state, a mixed disulfide between DsbD and the mutant thioredoxin was not detected (data not shown). Thus, similar to DsbC, the proposed disulfide intermediate between thioredoxin and DsbD appears to be unstable and may be reduced either by DsbD itself or by other cytoplasmic redox factors. To explore this possibility, cells expressing TrxA C35S and DsbD variants with single cysteine to alanine substitutions were analyzed by immunoblotting. Initial experiments failed to reveal a mixed disulfide between TrxA and DsbD. Perhaps some of the intermolecular disulfides formed by TrxA C35S are unstable and can be reduced by other factors in the E. coli cytoplasm.
We modified our experimental strategy and quenched all cellular disulfide exchange with MTSEA, a reagent that rapidly blocks cysteine thiols without attacking disulfides (32,33). When extracts of cells treated with MTSEA were analyzed by immunoblotting, DsbD C285A was observed to form an intermolecular disulfide with TrxA C35S (Fig. 3D) but not with wild-type TrxA (Fig. 3C). DsbD variants with alanine substitutions at any other cysteine did not produce this intermediate. MTSEA is a membrane-permeable methanethiosulfonate (33). Treatment of cells with MTSET, a membrane-impermeable methanethiosulfonate, did not stabilize the TrxA C35S -DsbD C285A disulfide (34). To identify the cysteine residue required for the formation of the mixed disulfide, we analyzed DsbD variants harboring alanine substitutions at cysteine 285 as well as at other cysteine residues. DsbD C163A/C285A failed to produce a mixed disulfide (Fig. 3D; lanes labeled C3 and C5 for DsbD C163A/C285A ), whereas alanine substitutions at other cysteines had no effect on the formation of the mixed disulfide (data not shown). DsbD and DsbD C285A were purified by affinity chromatography from MTSEA-treated cell extracts and examined by immunoblotting. Wild-type DsbD migrated as a single 56-kDa species on SDS-PAGE, whereas DsbD C285A appeared as a 56-and a 66-kDa species (Fig. 4). Antibodies raised against thioredoxin (␣-TrxA) bound to 66-kDa DsbD C285A but not to 56-kDa DsbD C285A or DsbD. DTT reduced the 66-kDa species to generate 56-kDa DsbD C285A and 10-kDa TrxA C35S (Fig. 4). Thus, electron transfer between TrxA and DsbD involves the formation of a disulfide bond between cysteine 32 of TrxA and cysteine 163 of DsbD. DISCUSSION Our results suggest a mechanism for the transfer of electrons across the cytoplasmic membrane of E. coli (Fig. 5). The NADPH cofactor of thioredoxin reductase donates electrons to reduce the disulfide within the active site dithiol motif of thi-oredoxin (29). Thioredoxin transfers its electrons directly to DsbD. Indeed, a mixed disulfide species could be captured between TrxA C35S -DsbD C285A . In this species, the thiol of cysteine 32 of TrxA is cross-linked to cysteine 163 in the DsbD FIG. 4. An intermolecular disulfide between DsbD C285A and TrxA C35S . DsbD or DsbD C285A , each carrying a C-terminal His 6 tag, were expressed together with TrxA C35S in E. coli strain A334. Cells were grown to A 595 nm 0.5 and incubated for 5 min at room temperature with 2.5 mM MTSEA to quench all disulfide bond rearrangements. Crude cellular extracts were subjected to affinity chromatography on nickel-nitrilotriacetic acid and eluted with imidazole. The eluate was incubated either with (ϩ) or without (Ϫ) 5 mM DTT, separated on 12% SDS-PAGE, and analyzed by immunoblotting. Proteins in lanes 1 and 2 as well as 6 and 7 were reacted with antibodies against DsbD (␣-DsbD), whereas proteins in lanes 3-5 and 8 -10 were reacted with antibodies against thioredoxin (␣-TrxA). Purified TrxA (lanes 5 and 10) was used as a control for the mobility of TrxA C35S on SDS-PAGE.

FIG. 3. Electron transfer between thioredoxin and DsbD involves the formation of an intermolecular disulfide.
A, E. coli strains expressing thioredoxin or various thioredoxin mutants were analyzed by trichloroacetic acid precipitation of proteins and immunoblotting with antibodies raised against DsbD (␣-DsbD). Wild-type thioredoxin (WT strain K38) harbors an active site dithiol motif (CXXC). However, the first cysteine residue (TrxA C32S , strain A333) or the second cysteine residue (TrxA C35S , strain A334) is substituted with serine in the mutant thioredoxin proteins. Strain A307 carries a deletion of the thioredoxin gene (⌬trxA). Treatment with AMS (12) slows the mobility of reduced DsbD compared with oxidized DsbD on SDS-PAGE. Both sulfhydryls of the active site dithiol of thioredoxin are needed to reduce oxidized DsbD. B, TrxA C35S , but not wild-type thioredoxin or TrxA C32S , generates intermolecular disulfide species with numerous other polypeptides as visualized by immunoblotting with ␣-TrxA. Plasmids encoding wild-type DsbD (DsbD ϩ ) as well as DsbD variants with alanine substitutions of single cysteine residues (C1-C7) were transformed into E. coli-expressing wild-type TrxA strain K38 (C) or TrxA C35S strain A334 (D). Cells were grown to A 595 nm 0.5 and incubated for 5 min at room temperature with 2.5 mM MTSEA to quench all disulfide bond rearrangements. Proteins were precipitated with trichloroacetic acid, separated on 8% SDS-PAGE, and analyzed by immunoblotting with ␣-DsbD. The position of DsbD and the intermolecular disulfide between DsbD and TrxA (C5) on SDS-PAGE is indicated by arrows. Alanine substitution at Cys 163 and Cys 285 of DsbD (C3 and C5) abrogated the formation of a mixed disulfide with TrxA C35S , whereas substitutions at Cys 285 and any other cysteine residue had no effect on the appearance of the intermolecular disulfide. molecule. We assume that during electron transfer a reversible disulfide bond is formed between cysteines 163 (C3) and 285 (C5) within the transmembrane region of DsbD. Upon reduction of this disulfide bond by TrxA, electrons will then travel within the DsbD molecule across the cytoplasmic membrane into the periplasm of E. coli. Reduction of cysteines 163 and 285 must be accompanied by a conformational change within DsbD. This conformational change likely allows the thiol of cysteine 285 to be approached by the disulfide in the C-terminal periplasmic domain of DsbD ( 461 CVAC). Even though such a reaction intermediate has not yet been demonstrated experimentally, we presume its existence because substitution of cysteine 461 (C6) with alanine abolishes electron transfer (25). Moreover, this defect is restored by expression of a soluble C-terminal periplasmic DsbD domain with intact cysteines (data not shown). In addition to bearing a typical thioredoxin motif (CXXC), this domain shares sequence similarity with the thioredoxin-like domain of protein-disulfide isomerase and thus is likely to adopt a thioredoxin fold (35) similar to proteindisulfide isomerase, DsbA, or DsbC (36 -38). Our results further predict that the C-terminal thioredoxin-like domain transfers its electrons to the N-terminal domain of DsbD by attacking the disulfide between cysteines 103 and 109 (C1 and C2). The final electron transfer step involves reduction of the DsbC dithiol motif ( 98 CGYC) via the formation of a disulfide intermediate with DsbD C109 . Although this has not yet been examined, electron transfer between DsbD and DsbE or DsbG, two periplasmic thiol oxidoreductases, presumably uses the same steps.
All electron transfer steps examined here occur by a common mechanism. The active site thiolate of the CXXC motif attacks disulfides, resulting in the formation of an intermolecular intermediate that is resolved by the second thiol. Electron transfer requires direct interaction between thioredoxin-like domains and unique peptide configurations harboring disulfides. Some of these interactions are promiscuous, e.g. recognition of the DsbD Cys 103 -Cys 109 disulfide by the dithiol motifs of DsbC, DsbE, and DsbG or interaction of DsbC, TrxA, and DsbA with folding substrates. However, the active site thiolate cannot resolve disulfides within another thioredoxin-like domain presumably because electrons cannot be favorably transferred to any one of these molecules. For example, DsbA does not recog-nize the DsbD Cys 103 -Cys 109 disulfide. This interaction would otherwise result in a wasteful flow of electrons between cytoplasmic, membrane, and periplasmic compartments. Oxidative protein folding catalyzed by the DsbA-DsbB system can be regarded as electron transfer from the periplasm to the membrane. It involves only two thioredoxin-like reactions: electrons flow from the substrate to the dithiol 30 (Fig. 5). Thus, it appears that DsbD has evolved as a fusion of two unique peptide configurations harboring disulfides tethered to a thioredoxin-like domain.
DsbD-mediated electron transfer across the cytoplasmic membrane of E. coli has been studied by several investigators (25,30,31). Stewart et al. (30) have examined the membrane topology of DsbD and were the first to propose a mechanism for sequential electron transfer. In the Stewart model, the thiolate within the C-terminal thioredoxin-like motif of DsbD attacks the disulfide of periplasmic DsbC, another thioredoxin-like motif. Electrons are transferred from the membrane-embedded disulfide of DsbD to the N-terminal disulfide and finally to the C-terminal periplasmic domain. Thioredoxin within the cytoplasm of E. coli attacks the membrane-embedded disulfide of DsbD and is recycled by thioredoxin reductase. It should be noted that the Stewart model was derived solely from topological analysis, positioning the cysteine residues of DsbD on either side of the cytoplasmic membrane; mixed disulfide species between interacting partners were not generated (30). As is demonstrated here, electrons flow from the N-terminal domain of DsbD to DsbC and not from the C-terminal domain of DsbD. Thus, the Stewart model does not appreciate the general mechanism of electron transfer between thioredoxin-like motifs and cognate peptides bearing disulfide bonds. In fact, step three of the Stewart model proposes transfer of electrons between the membrane-embedded disulfide of DsbD and the Nterminal periplasmic domain. As neither of these two domains assumes a thioredoxin-like motif, we believe that it is unlikely electrons can travel this path.
Our hypothesis presumes a conformational change of DsbD as the membrane-embedded disulfide/cysteines (Cys 163 and Cys 285 ) must be accessible to cytoplasmic thioredoxin and to the periplasmic domains of DsbD. If the distance between Cys 163 and Cys 285 were similar to the distance between the inner and outer leaflets of the cytoplasmic membrane, the conformational change would have to span a distance of 30 nm. Thus, disulfide bond formation would require mass movements of the polypeptide chain. We think it is more likely that DsbD assumes an overall conformation that minimizes the distance between Cys 163 and Cys 285 , allowing access of thioredoxin and of the C-terminal thioredoxin-like domain of DsbD within the plane of the cytoplasmic membrane. If so, electron transfer across the membrane may not require a massive conformational rearrangement and could make do with relatively small movements of DsbD transmembrane domains. Our future work will aim to characterize these events.