Previous Article | Next Article 
Journal of Bacteriology, February 2001, p. 980-988, Vol. 183, No. 3
Department of Chemical
Engineering,1 and Institute for Cell and
Molecular Biology,2 University of Texas, Austin,
Texas 78712; Department of Biology, University of Michigan, Ann
Arbor, Michigan 48109-10483; and
Department of Chemical Engineering, Stanford University,
Stanford, California 94305-50254
Received 15 September 2000/Accepted 20 November 2000
We have examined the role of the active-site CXXC central
dipeptides of DsbA and DsbC in disulfide bond formation and
isomerization in the Escherichia coli periplasm. DsbA
active-site mutants with a wide range of redox potentials were
expressed either from the trc promoter on a multicopy
plasmid or from the endogenous dsbA promoter by integration
of the respective alleles into the bacterial chromosome. The
dsbA alleles gave significant differences in the yield of
active murine urokinase, a protein containing 12 disulfides, including
some that significantly enhanced urokinase expression over that allowed
by wild-type DsbA. No direct correlation between the in vitro redox
potential of dsbA variants and the urokinase yield was
observed. These results suggest that the active-site CXXC motif of DsbA
can play an important role in determining the folding of multidisulfide
proteins, in a way that is independent from DsbA's redox potential.
However, under aerobic conditions, there was no significant difference
among the DsbA mutants with respect to phenotypes depending on the
oxidation of proteins with few disulfide bonds. The effect of
active-site mutations in the CXXC motif of DsbC on disulfide
isomerization in vivo was also examined. A library of DsbC expression
plasmids with the active-site dipeptide randomized was screened for
mutants that have increased disulfide isomerization activity. A number
of DsbC mutants that showed enhanced expression of a variant of human
tissue plasminogen activator as well as mouse urokinase were obtained.
These DsbC mutants overwhelmingly contained an aromatic residue at the
C-terminal position of the dipeptide, whereas the N-terminal residue
was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial
periplasmic space.
The formation of stable disulfide
bonds in gram-negative bacteria is catalyzed by the Dsb thiol-disulfide
oxidoreductase enzymes (30, 31). The oxidation of protein
thiols in newly secreted proteins is catalyzed by the periplasmic
enzyme DsbA. However, the formation of protein disulfide bonds by DsbA
occurs very rapidly and with little regard for the pairing of protein
cysteines in the native three-dimensional structure (41).
Disulfide bonds between cysteines that are not linked in the native
structure must be rearranged, a function that is catalyzed by two
homologous, homodimeric enzymes, DsbC and DsbG (8, 43).
Both enzymes are maintained in a reduced state in the periplasm through
the action of the integral membrane protein, DsbD, which transfers electrons from thioredoxin in the cytoplasm to the active site thiols
of DsbC and DsbG in the periplasmic space.
All the soluble Dsb proteins (DsbA, DsbC, and DsbG), as well as one
subdomain of the integral membrane protein DsbD, have been shown or
predicted to possess a common structural motif, the thioredoxin fold.
In the thioredoxin superfamily the catalytic cysteines are located
within a Cys-X-X-Cys motif. The two central amino acids in the
Cys-X-X-Cys motif strongly influence the intrinsic redox potential of
the active-site disulfide bond of thioredoxin family members
(12). Holmgren and coworkers initially showed that
substituting the dipeptide of Escherichia coli thioredoxin 1 with that found in eukaryotic protein disulfide isomerase (PDI) could
alter the redox properties of thioredoxin in the direction of PDI
(22, 23). The dipeptide was subsequently found to be important in determining the redox properties of many other members of
the thioredoxin family (11, 15, 18, 25, 26).
The identity of the active-site dipeptide in thiol-disulfide
oxidoreductases has also been shown to modulate oxidative protein folding in vivo in the eukaryotic endoplasmic reticulum and in the
bacterial periplasm (11, 15, 21). Grauschopf et al. constructed a library of DsbA mutants in which the active-site CXXC
dipeptide sequence had been randomized (15). This mutant library was screened for the degree of complementation of a
dsbA null mutant, using a modification of the screen
originally employed in the genetic isolation of the dsbA and
dsbB genes (38). Biochemical characterization
of these mutants showed that all had lower redox potentials than
wild-type DsbA. The pKa of the N-terminal cysteine of the
CXXC could be used to accurately predict the oxidizing power of each
mutant. However, no clear correlation between the redox potential of
the DsbA variants and the degree of complementation of a
dsbA null mutant could be discerned. More recently, variants of thioredoxin 1 (TrxA) expressed in the periplasm and containing the
active-site dipeptide normally found in DsbA, PDI, or the glutaredoxins
(containing, respectively, the sequence -PH-, -GH-, or -PY-) were shown
to partially alleviate two phenotypic defects in dsbA
mutants (21). Complementation was dependent on the
turnover of the more oxidizing TrxA variants by DsbB (13,
21). However, in the above-mentioned studies, the TrxA and DsbA
variants were expressed from multicopy plasmids at levels significantly
higher than the physiological levels of DsbA, thus making it difficult to discern effects resulting from protein concentration versus intrinsic catalytic properties.
In bacteria, disulfide isomerization represents the rate-limiting step
in the expression of complex eukaryotic proteins having multiple
disulfide bonds (27, 29). DsbC plays a major role in the
folding of multidisulfide proteins in the E. coli periplasm. For example, the formation of active mouse urokinase, which contains 12 disulfide bonds in its native state, is severely impaired in dsbC strains (33). Furthermore, human tissue
plasminogen activator having 17 disulfide bonds cannot be produced in
active form in E. coli unless DsbC is overexpressed at
moderately high levels (29). DsbA does not normally appear
to catalyze disulfide bond isomerization in vivo. Rather, it is
responsible for the formation of most of the aberrant disulfides in the
first place. Recent in vitro studies by Jonda et al. showed that the
catalytic proficiency of DsbA results in the formation of incorrect
disulfide bonds in hirudin. In contrast, catalysis of disulfide bond
formation by TrxA variants with less oxidizing redox potentials than
DsbA's resulted in higher yields of correctly folded hirudin
(21).
Given the significance of the active-site dipeptide on the redox
potential of thioredoxin superfamily members, we sought to examine its
effects on disulfide bond isomerization in vivo. Here we present a
systematic study on the effect of varying the active-site dipeptide on
the effectiveness of disulfide bond isomerization within model protein
substrates in the periplasm. We hypothesized that altering the
oxidizing power of DsbA and DsbC by mutagenesis might enhance the in
vivo folding of eukaryotic proteins that contain multiple disulfides.
We have succeeded in identifying several mutants of DsbA and DsbC that
significantly enhance the yield of multidisulfide proteins in the
periplasm. However, at least for the DsbA variants, the effect on the
folding of multidisulfide protein substrates showed no simple
correlation to the redox potential of the enzyme.
E. coli strains and plasmids.
The strains and
plasmids used in the present study are listed in Table
1. Plasmids containing the
dsbA mutant alleles are derivatives of pUG1
(15). The dsbA coding region was excised by
BamHI restriction digestion and cloned into pS1080 at the
unique BamHI site. The suicide vector pS1080 contains the
conditional R6K
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.3.980-988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effect of Sequences of the Active-Site Dipeptides
of DsbA and DsbC on In Vivo Folding of Multidisulfide Proteins in
Escherichia coli
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
origin and ampicillin resistance selectable marker,
as well as a counterselectable sacB gene, which confers
sucrose sensitivity. Allele exchange was conducted using the protocol
of Metcalf et al. (24) as modified by Bass et al.
(4). Cointegrates were transferred into the SF100
dsbA::kan strain background and
screened for loss of the kanr marker.
Following sucrose counterselection, colonies that were ampicillin
sensitive and sucrose resistant were screened by PCR using a forward
primer complementary to the native dsbA active-site sequence. Those colonies that failed to amplify, presumably due to
mismatches, were picked for further analysis. Allele exchange was
finally confirmed by amplification of the entire dsbA
reading frame followed by DNA sequencing. The
degP41::kan allele was then transduced
into the dsbA mutant strains using P1vir grown on SF110. The
unmarked 
degP allele in strain PB402 was generated by PCR amplification of the genomic flanking regions of degP and
ligation to create a deletion fragment in pS1080, which was used in
allele exchange as described above. Likewise, a strain carrying a
complete and unmarked deletion of dsbC (PB351) was
constructed in an analogous fashion.
TABLE 1.
Strains and plasmids used in this study
DsbC mutant library construction and screening.
Plasmid
pTrcdsbC was constructed as follows: the dsbC gene was
amplified from the chromosome with PCR primers dsbC.f and dsbC.b (Table
2). The resulting product was digested
with BspHI and HindIII and cloned into
pTrc99A (Amersham Pharmacia Biotech) at sites NcoI and
HindIII. Plasmid pBADdsbC(C118Stop) was constructed by
first amplifying plasmid pTrcdsbC by PCR using primers dsbC(C118Stop).f and dsbC.b. The resulting 390-bp product was used as the reverse primer
in a second reaction with the template pBADdsbC and forward primer
pBAD.s. The 830-bp product of the second reaction was digested with
XbaI and HindIII and cloned into pBADdsbC in
place of the dsbC coding region to generate
pBADdsbC(C118Stop). This plasmid contains DsbC with a mutation of
cysteine codon 118 (TGT) to the opal stop codon (TGA), a mutation of
cysteine codon 121 (TGC) to serine (AGC), and introduction of the
unique PmlI restriction site (CACGTG) directly 5' of the
active site by silent mutagenesis. Codon numbering is from the ATG
start codon of the open reading frame.
|
-D-thiogalactopyranoside (IPTG) to a final
concentration of 1 mM. After an additional 3 h of incubation at
30°C, the plates were removed and the turbidity at 595 nm was
measured. Cells were lysed by transferring 30 µl of the culture to
the wells of a plate containing 20 µl per well of cell lysis reagent,
BugBuster (Novagen), and shaking the plates for 30 min at room
temperature before freezing them at 
20°C. The plates were thawed at
room temperature, and the human tissue plasminogen activator (tPA)
activity of the lysate was measured by adding 200 µl of assay reagent
(0.01 µg of human Glu-type plasminogen [American Diagnostica,
Greenwich, Conn.] per µl, 0.1 mM Spectrozyme PL [American
Diagnostica] in 50 mM Tris-HCl [pH 7.4, at 37°C], 0.01% Tween
80). After incubation at 37°C for 90 min, the absorbance at 405 nm
was measured and normalized based on the optical density of the cells
at 595 nm as measured prior to lysis. Clones that produced amounts of
tPA similar to or greater than the amount produced by the positive
control (overexpressed wild-type DsbC) were retrieved from the
uninduced overnight plates and streaked onto solid media to isolate
single colonies. Plasmid DNA was isolated, and the region encoding the
dipeptide was sequenced using the primer pBAD.s. Subsequently, plasmids
were retransformed to DH5
, and colonies were selected that were
chloramphenicol resistant and carbenicillin sensitive, thus isolating
the DsbC plasmid away from the truncated-tPA (vtPA) plasmid. Plasmids
were prepared from the resulting colonies and used to retransform
competent DHB4/pTrcStIIvtPA cells. The control plasmids expressing
wild-type DsbC and truncated DsbC(C118Stop) were also transformed to
the fresh background at this time.
The effect of the DsbC mutant alleles was evaluated by growing the
cells in 125-ml shake flasks in 10 ml of LB medium with 50 µg of
carbenicillin per ml and 25 µg of chloramphenicol per ml at 30°C.
Cells were diluted 1:100 from an overnight culture, grown to an optical
density at 600 nm (OD600) of 0.8 and arabinose was added to
a final concentration of 0.2% to induce DsbC expression. Thirty
minutes later, vtPA expression was initiated by the addition of IPTG to
1 mM. After three additional hours of growth, the cells were pelleted
and frozen. Cell pellets were resuspended in cold phosphate-buffered
saline and lysed with a French press. Following the removal of
insoluble material by centrifugation (12,000 × g, 10 min at 4°C), the soluble protein concentration was determined by the
Bradford assay (Bio-Rad) using bovine serum albumin (BSA) as standard.
For tPA activity assays the lysates were diluted to 0.5 µg/µl in 50 mM Tris-HCl (pH 7.4 at 37°C)-0.01% Tween 80, and 10 µl of this
diluted lysate was added to 250 µl of the same buffer containing 0.04 µg of human Glu-type plasminogen per µl and 0.4 mM Spectrozyme PL.
To measure tPA activity, samples were incubated at room temperature,
and the A405 was monitored as a function of time.
Motility assay. Motility was assayed on M63 minimal salts soft agar (0.3%) supplemented with 18 amino acids (excluding cysteine and methionine), thiamine, and 0.2% glycerol. Overnight liquid cultures were diluted based on the optical density at 595 nm to normalize for cell number and were then inoculated into the center of a soft agar plate. After 24 h of incubation at 37°C, the diameter of the swarm was measured.
Enzymatic assays. To test the effect of various dsbA or dsbC variants on the yield of active urokinase, cells were first transformed with plasmid pRDB8-A or puPA184, each of which constitutively expresses secreted murine urokinase-type plasminogen activator. Overnight cultures in LB medium with 50 µg of carbenicillin per ml were diluted 1:100 into 5 ml of the same medium and grown at 30°C in test-tube cultures. In the uninduced DsbA experiments, the cultures were incubated for approximately 5 h, and the cells were pelleted and frozen. For coexpression of DsbC variants, the DsbC expression was induced by arabinose (final concentration, 0.2% [wt/vol]) at an OD595 of 0.8, and the cultures were grown for 3 additional h before being pelleted and frozen. Cell pellets were resuspended in 50 mM Tris-HCl (pH 7.4 at 37°C)-0.01% Tween 80, and lysed with a French press. Following the removal of insoluble material by centrifugation (12,000 × g, 10 min. at 4°C), the soluble protein concentration was determined by the Bradford assay (Bio-Rad) using BSA as the standard. Samples were diluted to 0.25 µg/µl in the buffer described above, and a 50-µl volume was added to a well containing 100 µl of the same buffer with 0.05 µg of human glu-type plasminogen per µl, 0.2 mM Spectrozyme PL, and 3 mM 6-amino-n-hexanoic acid. The plate was incubated at ambient temperature, and the absorbance at 405 nm was monitored.
Alkaline phosphatase (AP) activity was measured essentially as previously described (10) except that 1 mM iodoacetamide was included in all buffers to prevent spontaneous oxidation of reduced AP. Fibrin plate clearance assays were performed as described previously (29), using 10 µg of soluble cell lysate.| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Multicopy expression of DsbA active-site mutants.
Grauschopf and coworkers had constructed a library of
plasmid-encoded DsbA variants in which the active-site dipeptide
sequence was randomized (15). Mutants capable of
supporting the oxidation of the cysteines in a MalF--galactosidase
chimera, thus conferring a lacZ phenotype, were isolated.
The presence of the MalF moiety in MalF--galactosidase targets the
fusion protein to the membrane, and, as a result, a portion of
the -galactosidase portion is exported into the periplasm. In
cells expressing inactive or weakly active DsbA, the transmembrane
topology of MalF--galactosidase is unstable, and, therefore,
-galactosidase can retract into the cytoplasm where it is
enzymatically active, giving rise to blue colonies. In contrast, cells
expressing DsbA support the formation of disulfide bonds in the
periplasmic portion of -galactosidase which is thus misfolded and
inactive, resulting in the formation of white colonies.
Chromosomal integration of DsbA mutants. To distinguish between the influence of the active-site dipeptide sequence and the effect of the level of DsbA expression, we used allele replacement to transfer the set of 14 dsbA variants discussed above into the chromosome (Table 1). In addition, a mutant strain in which the two cysteines in the active site of the chromosomally encoded dsbA gene had been replaced with alanine (the APHA mutant) was also constructed. Allele replacements were confirmed by PCR amplification and DNA sequencing of the relevant region of the chromosome. In this manner, all the DsbA mutants were transcribed from the native promoter and translation was initiated from the wild-type ribosomal binding site.
In the study by Grauschopf et al., DsbA proteins having different active-site dipeptide sequences were ranked in terms of their oxidative capacity in vivo. The ranking was based on the ability of the DsbA variants to confer a lacZ phenotype on cells expressing MalF--galactosidase when grown in the presence of increasing
concentrations of the reductant dithiothreitol (DTT). The following
ranking of dipeptide sequences in terms of descending oxidative ability
in vivo was obtained (the central dipeptides are indicated by the
two-letter sequences): PH (wild type), PL, LQ > PR > PS, AL, QL,
SV > LT > ST > PP, TR, VL, SF, LL, QA, RC, FL.
According to the ranking, the least oxidizing mutants exhibited activities only slightly above background (dsbA)
(15). This ranking employed multicopy expression of DsbA
from an uninduced trc promoter (15). However,
Jonda et al. recently showed that overexpression of DsbA under some
conditions can lead to restored periplasmic protein oxidation and cell
motility, even in a dsbB mutant where the catalytic cycle of
DsbA cannot be completed (21). Therefore, it was of
interest to reexamine whether the DsbA mutants, when expressed from the
native promoter in the chromosome, could be ranked in a similar manner
on the basis of physiologically more relevant phenotypes.
Strains lacking DsbA exhibit slow growth in minimal salts media, both
in liquid and on agar plates. However, all the strains carrying
active-site dsbA alleles (PB420 to PB433) exhibited growth essentially indistinguishable from that of the corresponding parental strain carrying the wild-type gene (not shown). The motility of the
strains carrying the dsbA alleles was tested in minimal
media. Representative data are shown in Fig.
1. All of the active-site mutants,
regardless of their in vitro redox potentials, displayed motility
indistinguishable from that of the strain having the wild-type
dsbA allele, whether in a degP+ or
degP background. On the other hand, a dsbA mutant
and a dsbA(APHA) allele both displayed essentially no
motility, as expected.
|
220 mV (the PP mutant),
compared to 122 mV for the wild-type enzyme, nonetheless appear
completely normal with respect to two phenotypes dependent on the
oxidation of periplasmic proteins. The steady-state level of a protein
having one disulfide bond (-lactamase) was indistinguishable among
the dsbA alleles both in a degP+
background (strains PB420 to PB433) and in a degP background (strains PB401 to 419). In contrast, the formation of active murine urokinase was markedly affected by the sequence of the active-site dipeptide. This was especially evident with the chromosomal
dsbA alleles. For the chromosomal dsbA alleles,
urokinase activities ranged from negligible to significantly higher
than those of the parental strain (Fig.
2). Two of the less-oxidizing mutants, SF (152 mV) and PS (173 mV) displayed levels of urokinase activity that were three- to four-fold higher than those displayed by the wild-type strain, PH. However, mutants with similar redox potentials resulted in widely different levels of active urokinase (Fig. 2). Most
notably, the QL mutant that has an in vitro redox potential similar to
that of PS gave very low activity. The least-oxidizing mutant PP (220
mV) fell in the middle of the range in terms of the urokinase yield.
Likewise, the urokinase activity did not correlate with the
pKa of the reactive cysteine of the DsbA (15). Thus, it is clear that while the CXXC motif of DsbA strongly affects the ability of the cell to fold urokinase, it does so in a way that is
not directly related to the effects of these mutations on the redox
potential.
|
Effect of active-site dipeptide of DsbC on disulfide isomerization in vivo. As is the case with DsbA and all other proteins that have a thioredoxin fold, the active site of the bacterial disulfide isomerase, DsbC, contains a CXXC sequence. Presently there is no information on how the active-site dipeptide of DsbC affects the redox potential and the catalytic proficiency of the enzyme. To explore this issue, a library of DsbC mutants was constructed by randomizing the dipeptide sequence. This library was screened for clones that increase the yield of correctly folded heterologous proteins. No physiological substrates are known for DsbC, and null mutants do not exhibit any obvious phenotype. For this reason, the role of DsbC in catalyzing disulfide isomerization in vivo in E. coli was evaluated by its effect on the yield of heterologous proteins having multiple disulfides. These substrates include murine urokinase, bovine pancreatic trypsin inhibitor (BPTI) and human tissue plasminogen activator (29, 33, 44). Bessette et al. recently used a truncated form of tPA (vtPA) that consists of two (protease and kringle 2) of the five subdomains of the full-length protein and has nine disulfide bonds (7). When expressed in the bacterial periplasm, active vtPA accumulates to a very low level; however, cooverexpression of DsbC results in 100-fold higher yields of active protein. We decided to screen the DsbC library for mutants that upon overexpression result in a high yield of active vtPA for the following two reasons. (i) Given that a high intracellular amount of DsbC increases the yield of active vtPA, it is plausible that mutants with enhanced catalytic activity for disulfide bond formation and isomerization will result in even greater yields of active protein. This hypothesis was proven correct (see below). (ii) We have developed a convenient and sensitive assay for the yield of vtPA, thus enabling the screening of the DsbC library in a facile manner.
A library of DsbC mutants encoding a randomized active-site dipeptide sequence was constructed as described in Materials and Methods. Individual clones were grown in microtiter well plates to late exponential phase, the expression of DsbC and vtPA was induced for 3 h, and cells were lysed by a combination of chemical (nonionic detergent) and freeze-thaw lysis. Plasminogen activator activity was measured by a coupled chromogenic assay, and the results were normalized with respect to cell density. About 85% of the mutant clones were inactive in that they displayed no vtPA activity over background (i.e., cells without DsbC overexpression). Clones displaying a level of vtPA activity similar to (at least 90%) or greater than that displayed by the positive control were analyzed further. Plasmids were isolated, sequenced, and retransformed into fresh cells together with the vtPA expression vector. The cells were grown in shake flask cultures, and the DsbC mutants were ranked qualitatively based on the fibrin plate clearance assay (Table 3). For selected clones the vtPA level was also determined quantitatively, and the ranking was found to be consistent with the fibrin plate assay results (data not shown).
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The highly oxidizing state of the bacterial periplasm is
maintained primarily through the action of DsbA, which is the strongest oxidant of all known thiol-disulfide oxidoreductases. The redox potentials of the other two periplasmic oxidoreductases, DsbC and DsbG,
are quite comparable to that of DsbA (E0' for DsbC = 129 mV and E0' for DsbG = 124 mV [compare
E0' for DsbA = 122 mV]) (8, 39, 43).
Even though DsbC and DsbG can be oxidized by DsbA in vitro, in the cell
they are both maintained completely in a reduced state via the
DsbD-TrxA-TrxB pathway and the consumption of NADPH (8, 31,
33).
The redox potential of enzymes belonging to the thioredoxin superfamily
is critically dependent on the amino acid identity of the dipeptide
within the Cys-X-X-Cys active site (12, 18, 22, 23, 25). A
set of DsbA variants having different dipeptide sequences has been
previously isolated (15). When expressed in multicopy, and
in the presence of a reductant (DTT), these mutants support various
levels of oxidation of the -galactosidase moiety of a
MalF--galactosidase fusion. Fourteen of these dsbA mutants were transferred into the E. coli chromosome by
allele exchange. E. coli dsbA-null mutants are nonmotile,
grow poorly in minimal media, and express reduced levels of
periplasmic, disulfide-containing proteins such as -lactamase.
However, all the chromosomally encoded dsbA alleles fully
complemented these defects to a level comparable to that afforded by
the wild-type protein. Even DsbA variants poised at a very low redox
potential (e.g., for the CPPC active-site mutant, E0' = 220 mV) showed motility comparable to that of wild-type E. coli. These results suggest that the very high redox potential of
DsbA is not essential for many of the physiological functions of this
protein, at least under laboratory conditions. Most likely the
catalytic proficiency of DsbA becomes important in environments rich in
low-molecular-weight reductants. Along these lines, it should be noted
that the DsbA active-site dipeptide mutants analyzed in the present
study had been identified genetically on the basis of their ability to
oxidize a MalF--galactosidase fusion in the presence of increasing
concentrations of DTT (15). The more oxidizing DsbA
variants could oxidize the -galactosidase moiety of
MalF--galactosidase in cells grown with up to 3 mM DTT. Under these
conditions, less-oxidizing DsbA did not interfere with the folding of
active -galactosidase, resulting in the formation of blue colonies
(15). Although, the -galactosidase moiety of a
MalF--galactosidase fusion is not a physiological substrate for
DsbA, this result nonetheless demonstrates the importance of the
wild-type enzyme in mediating the formation of disulfide bonds in cells
grown in the presence of low-molecular-weight reductants.
Recently, Jonda et al. (21) reported that periplasmic targeting of TrxA variants with the CXXC sequence grafted from PDI, DsbA, or the glutaredoxins was able to partially restore the motility (and the lacZ) phenotype in a dsbA strain. These TrxA variants exhibit redox potentials comparable to or higher than that of the least-oxidizing DsbA variant examined in this study. The fact that the DsbA variants supported full complementation, while the TrxA variants did not, highlights the significance of kinetic considerations, in addition to the redox potential, with respect to disulfide bond formation in vivo.
While the DsbA mutants examined in this study had no obvious effect on
the oxidation of native periplasmic proteins, they exerted a rather
dramatic effect on the folding of a multidisulfide protein, murine
urokinase. The formation of active urokinase in E. coli is
critically dependent on the presence of both DsbA and DsbC in the
periplasm; no urokinase activity is evident in dsbA mutants,
and about 1% of the wild-type urokinase level is seen in
dsbC mutants (2, 32). All of the DsbA variants
having an altered active-site dipeptide increased the expression of
functional urokinase above background (dsbA). However, the
level of urokinase activity varied widely among the different mutants.
The activity increase could not be correlated directly with the redox
potential of DsbA. For example, the PS and QL mutants with essentially
the same in vitro redox potential (173 mV) gave urokinase activities differing by more than twenty-fold. Similarly, while the redox potentials of the SF and PL mutants have been experimentally determined as 152 mV and 156 mV, they gave, respectively, three-fold-higher and five-fold-lower active urokinase yields than the wild-type. These
differences could be observed because the dsbA alleles were expressed from a chromosomal copy. However, multicopy expression obscured the true effect of the DsbA active-site dipeptide mutants. For
example, in contrast to the results presented in Fig. 2, we found that
upon multicopy expression, the yields of murine urokinase obtained with
the CPLC mutant and wild-type DsbA were virtually indistinguishable.
Our data demonstrate that certain less-oxidizing DsbA mutants, while
sufficient for supporting normal cellular processes, are actually more
effective than the wild-type for the folding of proteins with several
disulfides. The wild-type DsbA is an extremely efficient protein thiol
oxidase and therefore causes the formation of aberrant disulfide bonds
in proteins such as BPTI and hirudin (three disulfides) in vitro
(40, 42). Jonda et al. have shown that the in vitro
catalyzed oxidative folding of hirudin using TrxA variants with redox
potentials between those of native TrxA (270 mV) and native DsbA
(122 mV) is more efficient than that afforded by DsbA
(21). Similarly, a circularly permuted DsbA variant with a
redox potential of 179 mV also showed improved folding of hirudin in
vitro (17). Interestingly, the refolding of hirudin with
the TrxA variants was five times faster than oxidation by wild-type
DsbA. Jonda et al. proposed that the TrxA variants promote the
consecutive formation of disulfide bonds which is more efficient than
the rapid, essentially random, thiol oxidation by DsbA. In the latter
case, extensive disulfide isomerization must take place before the
protein can reach the native state (21). However, the
situation in vivo is more complicated. When thiol oxidation is slow,
newly secreted polypeptide chains remain in a partially folded,
less-stable conformation longer and, as a result, they are more
vulnerable to degradation by the periplasmic proteolytic machinery
(28). A kinetic balance must be reached to attain the
maximum yield of multidisulfide proteins: oxidation needs to occur at a
rate fast enough to support the formation of proteolytically stable
intermediates but not so high that incorrect disulfide bonds prevent
correct folding. Evidently, the CPSC and CSFC mutants allow protein
oxidation to proceed at an optimal rate, resulting in greater protein
yield, at least for the murine urokinase substrate.
In addition to the effect of DsbA mutants, the formation of correctly folded multidisulfide proteins in E. coli is also greatly influenced by mutations in DsbC. A library of DsbC mutants with a randomized CXXC central dipeptide was screened for the ability to support the folding of a complex heterologous protein. The need to use a heterologous substrate was dictated by the fact that dsbC mutants exhibit no obvious phenotypes, and there are no specific physiological substrates that have been identified for this enzyme (32, 34). In contrast to the case of DsbA, where high-level expression can obscure the effect of mutations on disulfide bond isomerization, in the case of DsbC an elevated intracellular level of the enzyme (i.e., by high-level expression from a strong promoter), is essential for the formation of active vtPA in E. coli. For this reason, the library was screened for mutants that upon overexpression confer increased levels of vtPA activity. The majority of the clones analyzed (>85%) were inactive in the vtPA assay, indicating that only certain combinations of amino acids are tolerated within the active site. As can be seen in Table 3, a clear consensus for the sequence of the dipeptide emerged. The C-terminal amino acid in the active-site dipeptide (CXXC) exhibited a strong preference for hydrophobic amino acids and particularly for aromatic amino acids. Thirteen of 18 clones had phenylalanine, tyrosine, or histidine at the C-terminal position. Phenylalanine occurred at a much higher frequency (8 of 18) than the wild-type amino acid tyrosine (1 of 18). It is of interest to note that phenylalanine does not occur in that position in other known thioredoxin family members, except in the putative DsbC of certain Neisseria species, which contain the active site CPFC. The N-terminal position of the dipeptide (CXXC) was tolerant to a wider variety of amino acids, including basic, polar, and small hydrophobic residues. Acidic residues were not encountered in any of the clones that were scored in the screen.
Several mutants gave higher yields of murine urokinase and vtPA than the wild-type enzyme, indicating that they are at least equally, if not more, proficient in assisting the folding of multidisulfide proteins. The relative yields of vtPA and urokinase obtained with various DsbC variants was similar, indicating that the effect of the active-site mutations on disulfide bond isomerization is not limited to one polypeptide substrate. However, the precise molecular understanding of the effect of active-site mutations on the catalysis of disulfide bond isomerization will have to await detailed in vitro analyses of the equilibria and kinetics.
In summary, we have shown, for the first time, that mutations in the active sites of DsbA and DsbC can have a profound effect on the in vivo yield of multidisulfide proteins. The rate-limiting step in the folding of the proteins urokinase and vtPA in the E. coli periplasm appears to be the isomerization of incorrect disulfide bonds. The collection of dsbA alleles and the plasmids expressing DsbC mutants we have reported here are likely to be widely useful for aiding the periplasmic expression of multidisulfide proteins and for other biotechnology purposes.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by NSF GOALI 95-111 (G.G. and J.R.S.) and NIH 5RO1 GM55090-02. Paul Bessette was supported in part by an NIH Biotechnology Training Fellowship.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Chemical Engineering, University of Texas, Austin, TX 78712-1062. Phone: (512) 471-6975. Fax: (512) 471-7963. E-mail: gg{at}mail.che.utexas.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Baneyx, F., and G. Georgiou.
1990.
In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT.
J. Bacteriol.
172:491-494 |
| 2. |
Bardwell, J. C.,
J. O. Lee,
G. Jander,
N. Martin,
D. Belin, and J. Beckwith.
1993.
A pathway for disulfide bond formation in vivo.
Proc. Natl. Acad. Sci. USA
90:1038-1042 |
| 3. | Bardwell, J. C., K. McGovern, and J. Beckwith. 1991. Identification of a protein required for disulfide bond formation in vivo. Cell. 67:581-589[CrossRef][Medline]. |
| 4. |
Bass, S.,
Q. Gu, and A. Christen.
1996.
Multicopy suppressors of Prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated RlpA.
J. Bacteriol.
178:1154-1161 |
| 5. | Belin, D., J. D. Vassalli, C. Combepine, F. Godeau, Y. Nagamine, E. Reich, H. P. Kocher, and R. M. Duvoisin. 1985. Cloning, nucleotide sequencing and expression of cDNAs encoding mouse urokinase-type plasminogen activator. Eur. J. Biochem. 148:225-232[Medline]. |
| 6. | Bessette, P. H. 2000. Ph. D. dissertation. University of Texas at Austin, Austin. |
| 7. |
Bessette, P. H.,
F. Aslund,
J. Beckwith, and G. Georgiou.
1999.
Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm.
Proc. Natl. Acad. Sci. USA
96:13703-13708 |
| 8. |
Bessette, P. H.,
J. J. Cotto,
H. F. Gilbert, and G. Georgiou.
1999.
In vivo and in vitro function of the Escherichia coli periplasmic cysteine oxidoreductase DsbG.
J. Biol. Chem.
274:7784-7792 |
| 9. |
Boyd, D.,
C. Manoil, and J. Beckwith.
1987.
Determinants of membrane protein topology.
Proc. Natl. Acad. Sci. USA
84:8525-8529 |
| 10. | Brickman, E., and J. Beckwith. 1975. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J. Mol. Biol. 96:307-316[CrossRef][Medline]. |
| 11. | Chivers, P. T., M. C. Laboissiere, and R. T. Raines. 1996. The CXXC motif: imperatives for the formation of native disulfide bonds in the cell. EMBO J. 15:2659-2667[Medline]. |
| 12. | Chivers, P. T., K. E. Prehoda, and R. T. Raines. 1997. The CXXC motif: a rheostat in the active site. Biochemistry 36:4061-4066[CrossRef][Medline]. |
| 13. |
Debarbieux, L., and J. Beckwith.
2000.
On the functional interchangeability, oxidant versus reductant, of members of the thioredoxin superfamily.
J. Bacteriol.
182:723-727 |
| 14. | Duvoisin, R. M., D. Belin, and H. M. Krisch. 1986. A plasmid expression vector that permits stabilization of both mRNAs and proteins encoded by the cloned genes. Gene 45:193-201[CrossRef][Medline]. |
| 15. | Grauschopf, U., J. R. Winther, P. Korber, T. Zander, P. Dallinger, and J. C. Bardwell. 1995. Why is DsbA such an oxidizing disulfide catalyst? Cell 83:947-955[CrossRef][Medline]. |
| 16. |
Guzman, L. M.,
D. Belin,
M. J. Carson, and J. Beckwith.
1995.
Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.
J. Bacteriol.
177:4121-4130 |
| 17. | Hennecke, J., P. Sebbel, and R. Glockshuber. 1999. Random circular permutation of DsbA reveals segments that are essential for protein folding and stability. J. Mol. Biol. 286:1197-1215[CrossRef][Medline]. |
| 18. | Huber-Wunderlich, M., and R. Glockshuber. 1998. A single dipeptide sequence modulates the redox properties of a whole enzyme family. Fold Des. 3(3):161-171[CrossRef][Medline]. |
| 19. | Joly, J. C., and J. R. Swartz. 1997. In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC. Biochemistry 36:10067-10072[CrossRef][Medline]. |
| 20. | Joly, J. C., and J. R. Swartz. 1994. Protein folding activities of Escherichia coli protein disulfide isomerase. Biochemistry 33:4231-4236[CrossRef][Medline]. |
| 21. | Jonda, S., M. Huber-Wunderlich, R. Glockshuber, and E. Mössner. 1999. Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm. EMBO J. 18:3271-3281[CrossRef][Medline]. |
| 22. |
Krause, G.,
J. Lundstrom,
J. L. Barea,
C. Pueyo de la Cuesta, and A. Holmgren.
1991.
Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in Escherichia coli thioredoxin.
J. Biol. Chem.
266:9494-9500 |
| 23. |
Lundstrom, J.,
G. Krause, and A. Holmgren.
1992.
A Pro to His mutation in active site of thioredoxin increases its disulfide-isomerase activity 10-fold. New refolding systems for reduced or randomly oxidized ribonuclease.
J. Biol. Chem.
267:9047-9052 |
| 24. | Metcalf, W. W., W. Jiang, and B. L. Wanner. 1994. Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. Gene 138:1-7[CrossRef][Medline]. |
| 25. | Mössner, E., M. Huber-Wunderlich, and R. Glockshuber. 1998. Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases. Protein Sci. 7:1233-1244[Medline]. |
| 26. |
Mössner, E.,
M. Huber-Wunderlich,
A. Rietsch,
J. Beckwith,
R. Glockshuber, and F. Aslund.
1999.
Importance of redox potential for the in vivo function of the cytoplasmic disulfide reductant thioredoxin from Escherichia coli.
J. Biol. Chem.
274:25254-25259 |
| 27. |
Ostermeier, M.,
K. De Sutter, and G. Georgiou.
1996.
Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds.
J. Biol. Chem.
271:10616-10622 |
| 28. |
Ostermeier, M., and G. Georgiou.
1994.
The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm.
J. Biol. Chem.
269:21072-21077 |
| 29. |
Qiu, J.,
J. R. Swartz, and G. Georgiou.
1998.
Expression of active human tissue-type plasminogen activator in Escherichia coli.
Appl. Environ. Microbiol.
64:4891-4896 |
| 30. | Raina, S., and D. Missiakas. 1997. Making and breaking disulfide bonds. Annu. Rev. Microbiol. 51:179-202[CrossRef][Medline]. |
| 31. | Rietsch, A., and J. Beckwith. 1998. The genetics of disulfide bond metabolism. Annu. Rev. Genet. 32:163-184[CrossRef][Medline]. |
| 32. |
Rietsch, A.,
D. Belin,
N. Martin, and J. Beckwith.
1996.
An in vivo pathway for disulfide bond isomerization in Escherichia coli.
Proc. Natl. Acad. Sci. USA
93:13048-13053 |
| 33. |
Rietsch, A.,
P. Bessette,
G. Georgiou, and J. Beckwith.
1997.
Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin.
J. Bacteriol.
179:6602-6608 |
| 34. | Shevchik, V. E., G. Condemine, and J. Robert-Baudouy. 1994. Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemi and Escherichia coli with disulfide isomerase activity. EMBO J. 13:2007-2012[Medline]. |
| 35. | Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984. Experiments with gene fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 36. | Stafford, S. J., and P. A. Lund. 2000. Mutagenic studies on human protein disulfide isomerase by complementation of Escherichia coli dsbA and dsbC mutants. FEBS Lett. 466:317-322[CrossRef][Medline]. |
| 37. |
Strauch, K. L., and J. Beckwith.
1988.
An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins.
Proc. Natl. Acad. Sci. USA
85:1576-1580 |
| 38. |
Tian, H.,
D. Boyd, and J. Beckwith.
2000.
A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and sec machinery.
Proc. Natl. Acad. Sci. USA
97:4730-4735 |
| 39. | Wunderlich, M., and R. Glockshuber. 1993. Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli. Protein Sci. 2:717-726[Medline]. |
| 40. | Wunderlich, M., A. Otto, R. Seckler, and R. Glockshuber. 1993. Bacterial protein disulfide isomerase: efficient catalysis of oxidative protein folding at acidic pH. Biochemistry 32:12251-12256[CrossRef][Medline]. |
| 41. | Zapun, A., J. C. Bardwell, and T. E. Creighton. 1993. The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo. Biochemistry 32:5083-5092[CrossRef][Medline]. |
| 42. | Zapun, A., and T. E. Creighton. 1994. Effects of DsbA on the disulfide folding of bovine pancreatic trypsin inhibitor and alpha-lactalbumin. Biochemistry 33:5202-5211[CrossRef][Medline]. |
| 43. | Zapun, A., D. Missiakas, S. Raina, and T. E. Creighton. 1995. Structural and functional characterization of DsbC, a protein involved in disulfide bond formation in Escherichia coli. Biochemistry 34:5075-5089[CrossRef][Medline]. |
| 44. | Zhan, X., M. Schwaller, H. F. Gilbert, and G. Georgiou. 1999. Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase. Biotechnol. Prog. 15:1033-1038[CrossRef][Medline]. |
Journal of Bacteriology, February 2001, p. 980-988, Vol. 183, No. 3
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.3.980-988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Masip, L., Klein-Marcuschamer, D., Quan, S., Bardwell, J. C. A., Georgiou, G.
(2008). Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant. J. Biol. Chem.
283: 840-848
[Abstract] [Full Text] -
Quan, S., Schneider, I., Pan, J., Von Hacht, A., Bardwell, J. C. A.
(2007). The CXXC Motif Is More than a Redox Rheostat. J. Biol. Chem.
282: 28823-28833
[Abstract] [Full Text] -
Segatori, L., Murphy, L., Arredondo, S., Kadokura, H., Gilbert, H., Beckwith, J., Georgiou, G.
(2006). Conserved Role of the Linker {alpha}-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB. J. Biol. Chem.
281: 4911-4919
[Abstract] [Full Text] -
Elton, T. C., Holland, S. J., Frost, L. S., Hazes, B.
(2005). F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues. J. Bacteriol.
187: 8267-8277
[Abstract] [Full Text] -
Kim, J.-Y., Fogarty, E. A., Lu, F. J., Zhu, H., Wheelock, G. D., Henderson, L. A., DeLisa, M. P.
(2005). Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli. Appl. Environ. Microbiol.
71: 8451-8459
[Abstract] [Full Text] -
Tan, J., Lu, Y., Bardwell, J. C. A.
(2005). Mutational Analysis of the Disulfide Catalysts DsbA and DsbB. J. Bacteriol.
187: 1504-1510
[Abstract] [Full Text] -
Segatori, L., Paukstelis, P. J., Gilbert, H. F., Georgiou, G.
(2004). Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways. Proc. Natl. Acad. Sci. USA
101: 10018-10023
[Abstract] [Full Text] -
Horibe, T., Gomi, M., Iguchi, D., Ito, H., Kitamura, Y., Masuoka, T., Tsujimoto, I., Kimura, T., Kikuchi, M.
(2004). Different Contributions of the Three CXXC Motifs of Human Protein-disulfide Isomerase-related Protein to Isomerase Activity and Oxidative Refolding. J. Biol. Chem.
279: 4604-4611
[Abstract] [Full Text] -
Zhan, X., Gao, J., Jain, C., Cieslewicz, M. J., Swartz, J. R., Georgiou, G.
(2004). Genetic Analysis of Disulfide Isomerization in Escherichia coli: Expression of DsbC Is Modulated by RNase E-Dependent mRNA Processing. J. Bacteriol.
186: 654-660
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»