(Received for publication, April 17, 1995; and in revised form, May 31, 1995)
From the
Formation of disulfide bonds in Escherichia coli envelope proteins is facilitated by the Dsb system, which is
thought to consist of at least two components, a periplasmic soluble
enzyme (DsbA) and a membrane-bound factor (DsbB). Although it is
believed that DsbA directly oxidizes substrate cysteines and DsbB
reoxidizes DsbA to allow multiple rounds of reactions, direct evidence
for the DsbA-DsbB interaction has been lacking. We examined
intracellular activities of mutant forms of DsbA, DsbA30S and DsbA33S,
in which one of its active site cysteines (Cys
After translocation across the cytoplasmic membrane of
prokaryotic cells or the endoplasmic reticulum membrane of eukaryotic
cells, secretory proteins are folded into functional conformations. One
characteristic feature of the folding of secretory proteins is that
many of them must acquire correctly positioned disulfide
bonds(1) . Remarkable progress has been achieved recently in
our understanding of how protein disulfides are formed in the
periplasmic space of living prokaryotic cells. In Escherichia
coli, mutational inactivation of either dsbA(2, 3) , dsbB(4, 5) , or dsbC(6) gene
pleiotropically impairs disulfide bond formation of envelope proteins.
DsbA and DsbC are soluble proteins of the periplasm while DsbB is a
cytoplasmic membrane protein with four transmembrane
segments(7) . Among these proteins, the structure (8) and biochemical reactivities(9, 10, 11, 12, 13, 14, 15) of the
DsbA protein have been characterized in considerable details. It
directly oxidizes secretory proteins. The role of DsbB has been
proposed to reoxidize the reduced form of DsbA to enable it to work
again(4, 16) . Assuming the DsbA-DsbB circuit, it is
still unknown what are the acceptors of electrons that are obligatorily
liberated upon oxidation of the thiols. Little is known either about
DsbC, which, according to a proposal(16) , might be specialized
in isomerization of disulfide bonds.
The Dsb proteins all contain a
Cys-X-X-Cys motif seen characteristically for the
redox active sites in the thioredoxin-like
oxidoreductases(17) . The one in DsbA is
Cys
It was shown that mutational alterations of
either Cys
In this
work, we examined in vivo behaviors of the active site mutants
of DsbA that we constructed previously(13) . The results
indicate that the mutant form of DsbA (DsbA33S) in which Cys
Figure 1:
Complementing and interfering
activities of active site mutants of DsbA. Strain SS140 (dsbA::Tn5, lanes1-3) and
CU141 (dsbA
Figure 2:
DsbA33S is disulfide-bonded with other
proteins in the presence of small molecule disulfides. A,
cells of CU141 (dsbA
Figure 3:
Demonstration of a DsbA33S-DsbB complex. A, cells of CU141 (for all lanes except lane2) or SS141 (dsbB::kan5; lane2) that carried plasmids as indicated below were grown in
M9/glycerol medium and analyzed by non-reducing SDS-PAGE and
immunoblotting with anti-DsbA serum, as described in the legend to Fig. 2. Plasmids carried were: lanes1 and 2, pSS20 (dsbA33S); lane3, pSS18 (dsbA
Figure 4:
Suppression of the DsbA33S phenotypes by
GSSG. Cells as indicated below were grown in M9 medium supplemented
with GSSG at the final concentration of 0 (lanes 1-4), 1 (lanes 5-8), or 10 (lanes 9-12)
mM. Disulfide bond formation of Bla was examined by
non-reducing SDS-PAGE followed by immunoblotting. Strains used were: lanes 1, 5, and 9, CU141/pMW119 (vector); lanes
2, 6, and 10, SS140 (dsbA::Tn5)/pSS20 (dsbA33S); lanes 3, 7, and 11, CU141/pSS20; lanes 4, 8, and 12, SS141 (dsbB::kan5)/pMW119. It should be noted that the lower
bands in lanes 2-4 largely represent the background
protein of unknown identity (see also Fig. 1).
Like in any other thioredoxin-like oxidoreductases, the two
cysteine residues of DsbA are of vital importance for the physiological
functions of this enzyme. Detailed studies established that the two
cysteines have different reactivities. The amino-terminally located
Cys
It is shown here
that the DsbA33S mutant protein, in which only the reactive Cys
Although the formation of
disulfide-bonded complexes between DsbA33S and general protein
substrates requires the presence of GSSG or other small molecule
disulfides, DsbA33S is preferentially disulfide-bonded with DsbB in the
absence of GSSG. It should be noted that our method of detection, in
which cellular proteins were first denatured by trichloroacetic acid
and then dissolved in SDS in the presence of iodoacetamide, precludes
the possibility of artificial formation of the disulfide-bonded
complexes after cell disruption(21) . It seems likely that DsbA
has specific affinity to DsbB and that Cys
The high reactivity of Cys
We thank Jim Bardwell and Jon Beckwith for providing
the dsbB::kan5 mutant, Tohru Yoshihisa for the plasmid
carrying the Myc sequence and helpful comments and discussion,
Yoshinori Akiyama for helpful discussion throughout the work, and
Kiyoko Mochizuki and Kuniko Ueda for technical support.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or
Cys
, respectively) has been replaced by serine. The
DsbA33S protein was found to dominantly interfere with the disulfide
bond formation and to form intermolecular disulfide bonds with numerous
other proteins when cells were grown in media containing low molecular
weight disulfides such as GSSG. In the absence of added GSSG, DsbA33S
protein remained specifically disulfide-bonded with DsbB. These in
vivo results not only confirm the previous findings that
Cys
of DsbA is hyper-reactive in vitro but
provide evidence that DsbA indeed interacts selectively with DsbB. We
propose that the Cys
-mediated DsbA-DsbB complex represents
an intermediate state of DsbA-DsbB recycling reaction that has been
fixed because of the absence of Cys
on DsbA.
-Pro-His-Cys
. DsbB contains a set of
thioredoxin-like motifs as well as additional cysteine residues, among
which 4 essential Cys residues facing the periplasm have been
identified(7) .
or Cys
inactivate the enzymatic
activity of DsbA in vivo(18) or in
vitro(10, 13) . Cys
is exposed to
the solvent, is very reactive, and has a low pK
value, whereas Cys
is shielded from the
solvent(9, 10, 11, 12) .
was replaced by serine exhibited three unusual phenotypes. First,
it dominantly interferes with the functioning of the DsbA-DsbB system.
Second, it is disulfide-bonded with a number of cellular proteins when
cells are grown in the presence of small molecule disulfides such as
GSSG. Finally, it forms an intermolecular disulfide bond with DsbB
irrespective of the presence or absence of GSSG in the medium. We
suggest that DsbA preferentially interacts with DsbB, and the lack of
Cys
makes the interaction abortive with respect to the
catalytic turnover normally required for the functioning of the system.
Biological Materials
E. coli K12 strain
CU141 (19) was used as a dsb strain.
Strains SS140 and SS141 were CU141 derivatives into which the dsbA33::Tn5 mutation (2) or the dsbB::kan5 (4) mutation, respectively, had been
introduced by P1 transduction. Plasmid pSS18 carried dsbA
and was constructed by cloning the
1.0-kilobase EcoRI-HindIII fragment of the
pUC19-derived dsbA
plasmid (13) into
pMW119, a pSC101-based lac promoter vector (Nippon Gene).
Plasmids pSS19 and pSS20 were similar to pSS18, except for the presence
of the dsbA30S or the dsbA33S mutation (13) that caused a Cys
to Ser or a Cys
to Ser substitution in DsbA, respectively. pSS39 carried dsbB
; the dsbB region of the
chromosome was amplified by polymerase chain reaction using primers
5`AAACTGCGCACTCTATGC and 5`CGGGGATCCTTAGCGACCGAACAGATC, digested with NsiI and BamHI, and cloned into PstI-BamHI cleaved pSTV29 (a pACYC184-derived lac promoter vector from Takara Shuzo). pSS42 encoded a variant of
DsbB in which its carboxyl-terminal 10 amino acid residues had been
replaced by a sequence of 20 amino acids, IKLIDEEQKLISEEDLLRKR,
including a Myc epitope sequence; a 68-base pair EcoRV-KpnI fragment from a vector (pTYE006) carrying
a Myc-encoding sequence
(
)was inserted into
pSS39 that had been digested with DraI and KpnI. The
DsbB`-Myc protein retained the activity to complement the dsbB::kan5 mutation.
Analysis of the Formation of Intramolecular Disulfide
Bond in
Cells were grown at 37 °C either in peptone broth (20) or M9/glucose/amino acid medium (2) with or
without GSSG at a specified concentration. To overproduce products of
genes placed under the lac promoter control,
isopropyl--Lactamase and Intermolecular Mixed Disulfides Involving
DsbA
-D-thiogalactopyranoside was added to 2
mM. Bla
(
)was constitutively
synthesized from the plasmids. A portion of exponentially growing
cultures was mixed with an equal volume of 10% trichloroacetic acid to
denature and precipitate whole cell proteins, which were collected by
centrifugation (microcentrifuge for 2 min), washed with acetone, and
dissolved in 1% SDS, 1 mM EDTA, 50 mM Tris-HCl (pH
8.1) that contained 5 mM iodoacetamide. After separation by
SDS-PAGE in the absence of any reducing reagent(2) ,
polypeptides reactive with antiserum against Bla (5 Prime
3
Prime, Inc.), antiserum against DsbA(14) , or monoclonal
antibody against c-Myc (Ab-1, Oncogene Science, Inc.) were visualized
by immunoblotting (14) . In the case of the Myc antibody, the
second antibodies used were against mouse IgG.
DsbA33S Mutant Protein Exhibits Dominant Negative
Phenotype
Plasmids overexpressing either wild-type DsbA protein,
the DsbA30S mutant protein, or the DsbA33S mutant protein were
introduced into the dsbA-disrupted cells. Disulfide bond
formation was assessed by examining electrophoretic mobility of Bla,
which normally contains one intramolecular disulfide bond in SDS-PAGE
under non-reducing conditions. Consistent with the previous biochemical
results(10, 13) , both DsbA30S and DsbA33S were
inactive in supporting Bla oxidation in vivo (Fig. 1, lanes2 and 3). The same plasmids were then
introduced into wild-type cells. It was found that cells overproducing
DsbA33S were defective in disulfide bond formation of Bla (Fig. 1, lane6), indicating that DsbA with a
single cysteine at residue 30 is not only inactive but also interfering
with the normal functioning of the Dsb system.
, lanes 4-6) were
transformed with a plasmid carrying bla (for Bla) as well as
wild-type or mutant forms of dsbA as indicated below. Plasmids
used were: lanes1 and 4, pSS18 (dsbA
); lanes2 and 5, pSS19 (dsbA30S); lanes3 and 6, pSS20 (dsbA33S). Cells were grown in M9 medium.
After separation of whole cell proteins by SDS-PAGE under non-reducing
conditions, Bla was visualized by immunoblotting. red and ox indicate reduced and oxidized forms of Bla, respectively. A faintband that overlaps the lower edge of the
oxidized Bla is a nonspecific background.
DsbA33S Mutant Protein Is Disulfide-bonded with Other
Proteins in the Presence of Exogenously Added Small Molecule Disulfides
Fig. 2shows similar electropherograms as shown above,
but probed with anti-DsbA antibodies. In cells (with intact dsbA on the chromosome) overproducing DsbA33S, a number of higher
molecular mass bands were found to react with anti-DsbA (Fig. 2A, lane4). Most of these
higher molecular mass bands disappeared under reducing conditions (Fig. 2A, lane6), suggesting that
they represented mixed disulfides between DsbA and other proteins.
Results of non-reducing/reducing two-dimensional SDS-PAGE (data not
shown) suggested that the band of about 41 kDa (arrow in Fig. 2) represented a dimeric form of DsbA.(
)Cells overproducing DsbA
or DsbA30S
contained only a few cross-reacting proteins (Fig. 2A, lanes2 and 3), some of which (e.g. a band of about 33 kDa) were unrelated to the disulfide
cross-linking we were addressing since they persisted under the
reducing conditions (Fig. 2A, lanes5 and 6)
and they were observed even in the dsbA-disrupted cell (data not shown).
) that carried pMW119
(vector; lanes1 and 5), pSS18 (dsbA
; lane2), pSS19 (dsbA30S; lane3) or pSS20 (dsbA33S; lanes4 and 6) were grown
in peptone medium. B, cells of CU141/pSS20 (lanes1 and 3) and SS140 (dsbA::Tn5)/pSS20 (lanes2 and 4) were grown in M9 medium supplemented with (lanes3 and 4) or without (lanes1 and 2) 10 mM GSSG. In all cases, except for lanes5 and 6 of A, whole cell
proteins were separated by non-reducing SDS-PAGE, and proteins reactive
with anti-DsbA were visualized. Samples for lanes5 and 6 were separated after reduction with 5%
-mercaptoethanol. The arrow indicates a putative dimer of
DsbA,
and the arrowhead indicates the DsbA33S-DsbB
complex.
When cells were grown
on minimal salt medium rather than on broth, most of the higher
molecular mass cross-reacting polypeptides were not produced (Fig. 2B, lane1), with an exception
of a protein of about 36 kDa (arrowhead in Fig. 2).
Evidence presented in the following section shows that the 36-kDa
protein represents a DsbA33S-DsbB complex. The difference between the
broth and the salt media examined with respect to the production of the
cross-linked DsbA complexes appeared to be due to the presence of small
molecule disulfides such as cystine and GSSG in the former. This was
clearly demonstrated by the results presented in Fig. 2B, in which effects of GSSG addition to the M9
medium were examined. Thus, even in M9 medium, inclusion of GSSG
enhanced the formation of the intermolecular disulfide bonds involving
DsbA33S (Fig. 2B, compare lanes1 and 3). GSSG was effective at concentration of 1 mM (data
not shown) or 10 mM (Fig. 2B). It was found
additionally that the disulfide-mediated intermolecular cross-linking
was more extensive in the absence of chromosomal dsbA function than in its presence (Fig. 2B, compare lanes3 and 4).
DsbA33S Protein Preferentially Forms Mixed Disulfides
with DsbB
The apparent molecular weight of the major
DsbA33S-cross-linked polypeptide formed in minimal medium (without
GSSG) was similar to the sum of those of DsbA and DsbB. It indeed
represented a complex between DsbA33S and DsbB as the following lines
of evidence indicated. First, this particular product fractionated with
membranes (data not shown). Second, it was undetectable when DsbA33S
was overproduced in the dsbB::kan5 cells (Fig. 3A, compare lanes1 and 2). Third, when both DsbA33S and DsbB were overproduced, the
intensity of this product increased (Fig. 3A, compare lanes5 and 6). Finally, when a DsbB`-Myc
fusion protein that was 10 amino acids longer than the normal DsbB was
expressed from a plasmid, a new band of higher apparent molecular mass
was created (Fig. 3A, lane4). A band
that precisely coincided with this new band was stained by antibody
against the Myc epitope but only when DsbA33S was synthesized
simultaneously (Fig. 3B, lane2).
Two-dimensional SDS-PAGE (not shown) indicated that, upon reduction,
mobility of the Myc-cross-reacting material was shifted to that of
DsbB`-Myc (see Fig. 3B, lane1).(
)These results, taken
together, indicate that the DsbA33S protein specifically interacts with
DsbB to form disulfide-bonded heterodimers.
) and pSS42 (dsbB`-myc); lane4, pSS20 and pSS42; lane5,
pSS20 and pSTV29 (vector); lane6, pSS20 and pSS39 (dsbB
). The upper and lowerarrowheads indicate the DsbA33S-DsbB`-Myc complex and
DsbA33S-DsbB complex, respectively. B, the same samples as
used in lanes 3-6 of A were electrophoresed in lanes1-4, respectively, and stained with
anti-Myc antibody. The arrowhead indicates the
DsbA33S-DsB`-Myc complex, and the asterisk indicates the
DsB`-Myc protein.
Effects of GSSG on the Activities of DsbA33S Mutant
Protein
Previous studies showed that purified DsbA33S mutant
protein retains activities to bind glutathione as well as to reduce
oxidized insulin(10) . We also noted that the mutant form of
DsbA can stimulate oxidative folding of a mutant human lysozyme in the
presence of GSSG(13) . On the other hand, Bardwell et
al. (4) showed that GSSG phenotypically suppresses the dsbB disruption, probably by reoxidizing the reduced form of
DsbA. We examined effects of GSSG on the in vivo complementation and interference activities of DsbA33S. Inclusion
of 1 mM GSSG in the medium partially restored the
Bla-oxidizing ability of DsbA33S (Fig. 4, compare lanes2 and 6). GSSG at 1 mM almost
completely abolished the dominant negative effect of DsbA33S (Fig. 4, lane7). This concentration of GSSG
only slightly enhanced disulfide bond formation in the dsbB::kan5 strain (Fig. 4, lane8),
while 10 mM GSSG effectively suppressed the dsbB mutation (Fig. 4, lane12). GSSG up to 10
mM did not restore the defective disulfide bond formation in
the dsbA-disrupted strain ((4) ),(
)indicating that GSSG can help the functioning of
DsbA in the absence of DsbB but cannot substitute for the DsbA function
itself(4) . The effective suppression of the dominant
interference by 1 mM GSSG might be due to activation of the
DsbA33S form of DsbA. Such an unusual mode of action of DsbA should be
independent of the DsbB function.
has unusually low pK
(12) and is very reactive with alkylating reagents
as well as with small molecule sulfhydryls(9, 10) .
The mixed disulfide between GSH and Cys
of DsbA is very
unstable(10) . Although similar instability has been noted for
the oxidized form of wild-type DsbA (9, 10, 11) , DsbA in the periplasmic space
of living cells is mostly in the oxidized state, provided that DsbB is
functional(5, 21) . In contrast to Cys
,
Cys
residue is buried inside the protein and inaccessible
from the solvent(9, 10, 12) . Apparently, the
asymmetric nature of the two cysteines contributes to the catalytic
reactivity of this unusual enzyme(16) .
residue remains, is disulfide-bonded with a number of cellular
proteins in the presence of low molecular weight disulfides. It might
be possible that the reactive Cys
residue first forms
mixed disulfides with cystine or glutathione added exogenously. This
binding may activate the protein. Due possibly to the
polypeptide-binding nature of DsbA(8) , the disulfide bond may
be then transferred to a free cysteine on the substrate protein.
Repeating this cycle might lead to the formation of protein disulfides
in the presence of GSSG(13) .
of DsbA can
react with one of the four essential cysteines of DsbB without
mediation of GSSG.
of DsbA
with a cysteine residue of DsbB will contribute to the dominant
interference by the DsbA33S mutant protein, which should sequester the
DsbB protein in the heterodimeric complex. More importantly, the high
reactivity of the cysteine residues on the DsbA and the DsbB protein
will contribute to the proposed (4, 16) reoxidation of
the reduced form of DsbA by DsbB. We suppose it likely that the
DsbA33S-DsbB complex we observed here represents an intermediate state
of DsbA-DsbB recycling reaction that has been fixed because of the
absence of the partner cysteine, Cys
, on DsbA.
-lactamase; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.