DsbD-catalyzed Transport of Electrons across the Membrane of Escherichia coli*

Rebecca Krupp, Cecilia Chan, and Dominique MissiakasDagger

From the Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 90095

Received for publication, October 18, 2000, and in revised form, November 14, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 98CGYC. 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 98CGYC101 thiol (sulfhydryl) residues of DsbC are oxidized (DsbC-S2). 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-S2) 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 µg/ml; spectinomycin, 50 µg/ml; chloramphenicol, 20 µg/ml; and kanamycin, 50 µg/ml. Induction of His6-tagged DsbD using the pSE420 plasmid (Invitrogen) was accomplished by addition of isopropyl-1-thio-beta -D-galactopyranoside to a final concentration of 1 mM. Plasmids expressing TrxAC32S (pTrxAC32S) and TrxAC35S (pTrxAC35S) were obtained from M. Russel (Rockefeller University). Plasmids pRK35 and pCC68 expressing wild-type DsbC and DsbCC101A 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).


                              
View this table:
[in this window]
[in a new window]
 
Table I
Bacterial strains and plasmids

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 His6 tag, His6-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 (MTSEA) or 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) reagents. Cells were grown to A595 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 -80 °C until further use. The redox state of DsbD in vivo was assessed using AMS as described previously (12).

Protein Purification-- Wild-type DsbD (pJC350), DsbDC103A (pCC61), and DsbDC285A (pCC15) with an appended C-terminal His6 tag were expressed by cloning the structural genes into pSE420 (Invitrogen). Cells expressing either one of the various His6-tagged DsbD proteins were grown to A595 nm and incubated for 2 h with 1 mM isopropyl-1-thio-beta -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 × 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 His6-tagged DsbD proteins on nickel-nitrilotriacetic acid.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (135CATC138) and one FAD molecule and catalyzes the reduction of TrxA by NADPH (22, 28). TrxA is a small cytoplasmic protein (~10 kDa) that contains one redox active site 32CGPC35 (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 Cys135 attacks the disulfide of TrxA-S2 to form transiently the mixed disulfide between TrxB and TrxA, Cys135-Cys32. This mixed intermediate is resolved by the Cys138 thiolate, resulting in the release of reduced TrxA-(SH2) (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 Cys138 of TrxB and Cys35 of TrxA for serine captures the mixed disulfide between TrxB and TrxA, the C135-C32 intermediate (29). Similarly, substitution of the second cysteine (Cys33) of the dithiol of DsbA with alanine (30CPHA) leads to the production of a stable complex with a disulfide bond between Cys30 of DsbA and Cys104 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 (DsbCC101A) or TrxA (TrxAC35S) 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 ~10 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, Cys103-Cys109 and Cys461-Cys464, respectively (Fig. 1A). The two domains are connected by eight transmembrane (TM) segments containing three cysteine residues, Cys163 (TM1), Cys282 (TM4), and Cys285 (TM4) (Fig. 1A). DsbD residue Cys282 (C4) is dispensable for electron transfer. In contrast, alanine substitution of cysteine residues at any one of the other cysteines (C103 (C1), Cys109 (C2), Cys163 (C3), Cys285 (C5), Cys461 (C6), and Cys464 (C7)) abolishes DsbD-mediated electron transfer, causing accumulation of DsbC-S2 (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, Cys101 of DsbC.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Electron transfer between DsbC and DsbD involves the formation of an intermolecular disulfide. A, the drawing illustrates the membrane topology of DsbD. The N- and C-terminal periplasmic domains are connected by eight transmembrane domains (roman numerals). The position of cysteine sulfhydryl residues are indicated: C1 (Cys103), C2 (Cys109), C3 (Cys163), C4 (Cys282), C5 (Cys285), C6 (Cys461), and C7 (Cys464). Plasmids encoding wild-type DsbD (dsbD+) (B) and plasmids encoding DsbD variants with alanine substitutions of single cysteine residues (C1-C7) (C) were transformed into E. coli-expressing DsbCC101A or wild-type DsbC. Proteins were precipitated with trichloroacetic acid, separated on SDS-PAGE, and analyzed by immunoblotting with alpha -DsbD. The position of DsbD and the intermolecular disulfide between DsbD and DsbC (C1) on SDS-PAGE is indicated by arrows. Alanine substitution at Cys103 and Cys109 of DsbD (C1 and C2) abrogated mixed disulfide formation with DsbC, whereas substitutions at Cys103 and any other cysteine residue had no effect on the appearance of the intermolecular disulfide (data not shown).

To test this possibility, E. coli strains expressing DsbCC101A and various DsbD cysteine substitution mutants were examined. An intermolecular intermediate was observed in cells expressing DsbCC101A and DsbDC103A (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 intermolecular intermediate, DsbDC103A with a C-terminal His6 tag was purified from E. coli extracts by affinity chromatography (Fig. 2). Immunoblotting of eluted fractions revealed the presence of two electrophoretic species with an apparent molecular mass of 56 (DsbDC103A, alone) and 80 kDa (DsbDC103A-DsbCC101A), respectively. Antibodies raised against DsbD (alpha -DsbD) and DsbC (alpha -DsbC) recognized the 80-kDa DsbDC103A-DsbCC101A species. In contrast, only alpha -DsbD bound to 56-kDa DsbDC103A. 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). alpha -DsbC, but not alpha -DsbD, bound to the 24-kDa protein, indicating that this species represents DsbCC101A released from its disulfide linkage with DsbDC103A. Cysteine 98 of DsbC, the first sulfhydryl within the dithiol motif (98CGYC), must be one component of the intermolecular disulfide with DsbD. To identify the corresponding cysteine residue of DsbD, we analyzed DsbD variants harboring alanine substitutions at Cys103 as well as at other cysteine residues. DsbDC103A/C109A failed to form the intermolecular disulfide with DsbCC101A (Fig. 1C; lanes labeled C1 and C2 for DsbDC103A/C109A). All other combinations of alanine substitutions produced the mixed disulfide species (data not shown). Thus, electron transfer between DsbD and DsbC involves the formation of a disulfide between cysteine 109 of DsbD and cysteine 98 of DsbC. Further, the intermolecular disulfide is resolved by the Cys103 thiol of DsbD, generating the intramolecular cystine Cys103-Cys109 (C1 and C2) as well as reduced DsbC-(SH)2.



View larger version (70K):
[in this window]
[in a new window]
 
Fig. 2.   An intermolecular disulfide between DsbDC103A and DsbCC101A. DsbDC103A with a C-terminal His6 tag was expressed together with DsbCC101A in the E. coli strain DM2441 (K38 Delta dsbD Delta dsbC). Crude cellular extracts were subjected to affinity chromatography on nickel-nitrilotriacetic acid and eluted with imidazole. The eluate was incubated either with (+) or without (-) 5 mM DTT, separated on 12% SDS-PAGE, and analyzed by immunoblotting. Proteins in lanes 1 and 2 were reacted with antibodies against DsbD (alpha -DsbD), whereas proteins in lanes 3-5 were reacted with antibodies against DsbC (alpha -DsbC). Purified DsbC (lane 5) was used as a control for the mobility of DsbCC101A on SDS-PAGE.

Electron Transfer between TrxA and DsbD-- E. coli lacking thioredoxin (Delta trxA) accumulate oxidized DsbC (20, 25) and DsbD (Fig. 3A). Both cysteines of the dithiol motif of thioredoxin (32CGPC35) are required for electron transfer, as either mutant TrxAC32S or mutant TrxAC35S accumulated oxidized DsbD (Fig. 3A). Substitution of TrxAC35S but not TrxAC32S produced a spectrum of disulfide-linked intermolecular intermediates (Fig. 3B). Thus, substitution of the second cysteine of thioredoxin with serine (TrxAC35S) results in a mutant protein capable of attacking numerous cytoplasmic disulfides but unable to resolve the mixed disulfide intermediates. Although cells expressing TrxAC35S 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 TrxAC35S 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 TrxAC35S are unstable and can be reduced by other factors in the E. coli cytoplasm.



View larger version (45K):
[in this window]
[in a new window]
 
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 (alpha -DsbD). Wild-type thioredoxin (WT strain K38) harbors an active site dithiol motif (CXXC). However, the first cysteine residue (TrxAC32S, strain A333) or the second cysteine residue (TrxAC35S, strain A334) is substituted with serine in the mutant thioredoxin proteins. Strain A307 carries a deletion of the thioredoxin gene (Delta 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, TrxAC35S, but not wild-type thioredoxin or TrxAC32S, generates intermolecular disulfide species with numerous other polypeptides as visualized by immunoblotting with alpha -TrxA. Plasmids encoding wild-type DsbD (DsbD+) 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 TrxAC35S strain A334 (D). Cells were grown to A595 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 alpha -DsbD. The position of DsbD and the intermolecular disulfide between DsbD and TrxA (C5) on SDS-PAGE is indicated by arrows. Alanine substitution at Cys163 and Cys285 of DsbD (C3 and C5) abrogated the formation of a mixed disulfide with TrxAC35S, whereas substitutions at Cys285 and any other cysteine residue had no effect on the appearance of the intermolecular disulfide.

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, DsbDC285A was observed to form an intermolecular disulfide with TrxAC35S (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 TrxAC35S-DsbDC285A 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. DsbDC163A/C285A failed to produce a mixed disulfide (Fig. 3D; lanes labeled C3 and C5 for DsbDC163A/C285A), whereas alanine substitutions at other cysteines had no effect on the formation of the mixed disulfide (data not shown). DsbD and DsbDC285A 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 DsbDC285A appeared as a 56- and a 66-kDa species (Fig. 4). Antibodies raised against thioredoxin (alpha -TrxA) bound to 66-kDa DsbDC285A but not to 56-kDa DsbDC285A or DsbD. DTT reduced the 66-kDa species to generate 56-kDa DsbDC285A and 10-kDa TrxAC35S (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.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 4.   An intermolecular disulfide between DsbDC285A and TrxAC35S. DsbD or DsbDC285A, each carrying a C-terminal His6 tag, were expressed together with TrxAC35S in E. coli strain A334. Cells were grown to A595 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 (+) or without (-) 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 (alpha -DsbD), whereas proteins in lanes 3-5 and 8-10 were reacted with antibodies against thioredoxin (alpha -TrxA). Purified TrxA (lanes 5 and 10) was used as a control for the mobility of TrxAC35S on SDS-PAGE.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 thioredoxin (29). Thioredoxin transfers its electrons directly to DsbD. Indeed, a mixed disulfide species could be captured between TrxAC35S-DsbDC285A. In this species, the thiol of cysteine 32 of TrxA is cross-linked to cysteine 163 in the DsbD 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 (461CVAC). 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 protein-disulfide 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 (98CGYC) via the formation of a disulfide intermediate with DsbDC109. Although this has not yet been examined, electron transfer between DsbD and DsbE or DsbG, two periplasmic thiol oxidoreductases, presumably uses the same steps.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Model for DsbD-mediated transfer of electrons across the cytoplasmic membrane. TrxB (NADPH) donates electrons to the active site dithiol motif of TrxA. TrxA resolves the disulfide between cysteines 163 and 285 of DsbD (C3 and C5). Cysteines 163 and 285 donate electrons to the thioredoxin motif of DsbD thereby reducing the dithiol at cysteines 461 and 464 (C6 and C7). The reduced dithiol attacks the disulfide at cysteines 103 and 109 (C1 and C2), which transfer electrons to the oxidized dithiol motif of DsbC. The square-shaped box indicates the presence of a thioredoxin-like fold and dithiol motif. Electron transfer occurs as a sequence of disulfide rearrangements between cysteine pairs arranged in a thioredoxin-like motif (CXXC) with a cognate reactive disulfide.

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 Cys103-Cys109 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 recognize the DsbD Cys103-Cys109 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 30CPHC of DsbA to DsbB Cys104-C130 disulfide and then to the dithiol 41CVLC of DsbB and finally to the electron transport system (39). In contrast, electron transfer from the cytoplasm to the periplasm requires a sequence of three thioredoxin-like reactions: 1) [Cys32]TrxA-[Cys163]DsbD, 2) [Cys285]DsbD-[Cys461]DsbD, and 3) [Cys109]DsbD-[Cys98]DsbC (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 N-terminal 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 (Cys163 and Cys285) must be accessible to cytoplasmic thioredoxin and to the periplasmic domains of DsbD. If the distance between Cys163 and Cys285 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 Cys163 and Cys285, 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.


    ACKNOWLEDGEMENTS

We thank O. Schneewind (UCLA) for critical review of this manuscript.


    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Microbiology, Immunology, and Molecular Genetics, UCLA, 609 Charles Young Dr., Los Angeles, CA 90095. Tel.: 310-794-9395; Fax: 310-267-0173; E-mail: missiaka@microbio.ucla.edu.

Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M009500200


    ABBREVIATIONS

The abbreviations used are: TrxA, thioredoxin; TrxB, thioredoxin reductase; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; DTT, dithiothreitol.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Creighton, T. E. (1993) Proteins , 2nd Ed. , W. H. Freeman and Company, New York
2. Freedman, R. B. (1989) Cell 57, 1069-1072[Medline] [Order article via Infotrieve]
3. Frand, A. R., Cuozzo, J. W., and Kaiser, C. A. (2000) Trends Cell Biol. 10, 203-210[CrossRef][Medline] [Order article via Infotrieve]
4. Raina, S., and Missiakas, D. (1997) Annu. Rev. Microbiol. 51, 179-202[CrossRef][Medline] [Order article via Infotrieve]
5. Rietsch, A., and Beckwith, J. (1998) Annu. Rev. Genet. 32, 163-184[CrossRef][Medline] [Order article via Infotrieve]
6. Bardwell, J. C., McGovern, K., and Beckwith, J. (1991) Cell 67, 581-589[Medline] [Order article via Infotrieve]
7. Kamitani, S., Akiyama, Y., and Ito, K. (1992) EMBO J. 11, 57-62[Abstract]
8. Bardwell, J. C. A., Lee, J.-O., Jander, G., Martin, N., Belin, D., and Beckwith, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1038-1042[Abstract]
9. Missiakas, D., Georgopoulos, C., and Raina, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7084-7088[Abstract]
10. Guilhot, C., Jander, G., Martin, N. L., and Beckwith, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9895-9899[Abstract]
11. Kishigami, S., Kanaya, E., Kikuchi, M., and Ito, K. (1995) J. Biol. Chem. 270, 17072-17074[Abstract/Free Full Text]
12. Kobayashi, T., Kishigami, S., Sone, M., Inokuchi, H., Mogi, T., and Ito, K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11857-11862[Abstract/Free Full Text]
13. Bader, M., Muse, W., Ballou, D. P., Gassner, C., and Bardwell, J. C. (1999) Cell 98, 217-227[Medline] [Order article via Infotrieve]
14. Zapun, A., Missiakas, D., Raina, S., and Creighton, T. E. (1995) Biochemistry 34, 5075-5089[Medline] [Order article via Infotrieve]
15. Missiakas, D., Georgopoulos, C., and Raina, S. (1994) EMBO J. 13, 2013-2020[Abstract]
16. Fabianek, R. A., Hennecke, H., and Thony-Meyer, L. (1998) J. Bacteriol. 180, 1947-1950[Abstract/Free Full Text]
17. Andersen, C. L., Matthey-Dupraz, A., Missiakas, D., and Raina, S. (1998) Mol. Microbiol. 26, 121-132
18. Bessette, P. H., Cotto, J. J., Gilbert, H. F., and Georgiou, G. (1999) J. Biol. Chem. 274, 7784-7792[Abstract/Free Full Text]
19. Missiakas, D., Schwager, F., and Raina, S. (1995) EMBO J. 14, 3415-3424[Abstract]
20. Rietsch, A., Bessette, P., Georgiou, G., and Beckwith, J. (1997) J. Bacteriol. 179, 6602-6608[Abstract]
21. Russel, M. (1995) Methods Enzymol. 252, 264-274[Medline] [Order article via Infotrieve]
22. Holmgren, A. (1995) Structure 3, 239-243[Medline] [Order article via Infotrieve]
23. Miller, J. (1992) A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Proteins , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
24. Ansaldi, M., Lepelletier, M., and Mejean, V. (1996) Anal. Biochem. 234, 110-111[CrossRef][Medline] [Order article via Infotrieve]
25. Chung, J., Chen, T., and Missiakas, D. (2000) Mol. Microbiol. 35, 1099-1109[CrossRef][Medline] [Order article via Infotrieve]
26. Wang, R. F., and Kushner, S. R. (1991) Gene (Amst.) 100, 195-199[CrossRef][Medline] [Order article via Infotrieve]
27. Rietsch, A., Belin, D., Martin, N., and Beckwith, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13048-13053[Abstract/Free Full Text]
28. Lennon, B. W., Williams, C. H. J., and Ludwig, M. L. (2000) Science 289, 1190-1194[Abstract/Free Full Text]
29. Wang, P., Veine, D. M., Ahn, S. H., and Williams, C. H. J. (1996) Biochemistry 35, 4812-4819[CrossRef][Medline] [Order article via Infotrieve]
30. Stewart, E. J., Katzen, F., and Beckwith, J. (1999) EMBO J. 18, 5963-5971[Abstract/Free Full Text]
31. Gordon, E. H., Page, M. D., Willis, A. C., and Ferguson, S. J. (2000) Mol. Microbiol. 35, 1360-1374[CrossRef][Medline] [Order article via Infotrieve]
32. Smith, D. J., Maggio, E. T., and Kenyon, G. L. (1975) Biochemistry 14, 764-771
33. Akabas, M. H., and Karlin, A. (1995) Biochemistry 34, 12496-12500[Medline] [Order article via Infotrieve]
34. Stauffer, D. A., and Karlin, A. (1994) Biochemistry 33, 6840-6849[Medline] [Order article via Infotrieve]
35. Ellis, L. B., Saurugger, P., and Woodward, C. (1992) Biochemistry 31, 4882-4891[Medline] [Order article via Infotrieve]
36. Kemmink, J., Darby, N. J., Dijkstra, K., Nilges, M., and Creighton, T. E. (1997) Curr. Biol. 7, 239-245[Medline] [Order article via Infotrieve]
37. Martin, J. L., Bardwell, J. C., and Kuriyan, J. (1993) Nature 365, 464-468[CrossRef][Medline] [Order article via Infotrieve]
38. McCarthy, A. A., Haebel, P. W., Torronen, A., Rybin, V., Baker, E. N., and Metcalf, P. (2000) Nat. Struct. Biol. 7, 196-199[CrossRef][Medline] [Order article via Infotrieve]
39. Debarbieux, L., and Beckwith, J. (1999) Cell 99, 117-119[Medline] [Order article via Infotrieve]
40. Russel, M., Model, P., and Holmgren, A. (1990) J. Bacteriol. 172, 1923-1929[Medline] [Order article via Infotrieve]
41. Takeshita, S., Sato, M., Toba, M., Masahashi, W., and Hashimoto-Gotoh, T. (1987) Gene 61, 63-74[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.