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
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ABSTRACT |
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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.
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.
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- 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 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- 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.
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 ( Electron Transfer between TrxA and DsbD--
E. coli
lacking thioredoxin (
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 ( 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
Bacterial strains and plasmids
80 °C until further use. The
redox state of DsbD in vivo was assessed using AMS as
described previously (12).
-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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 -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).
-DsbD) and DsbC (
-DsbC)
recognized the 80-kDa DsbDC103A-DsbCC101A
species. In contrast, only
-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).
-DsbC, but not
-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.
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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 dsbD
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 (
-DsbD), whereas proteins in lanes 3-5 were
reacted with antibodies against DsbC (
-DsbC). Purified DsbC
(lane 5) was used as a control for the mobility of
DsbCC101A on SDS-PAGE.
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.
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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 (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 (
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
-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
-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.
-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.
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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
(
-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 TrxAC35S on SDS-PAGE.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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ACKNOWLEDGEMENTS |
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We thank O. Schneewind (UCLA) for critical review of this manuscript.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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