(Received for publication, April 7, 1997, and in revised form, June 19, 1997)
From the Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, CH-8093 Zürich, Switzerland
Disulfide oxidoreductases are structurally
related proteins that share the thioredoxin fold and a catalytic
disulfide bond that is located at the N terminus of an -helix. The
different redox potentials of these enzymes varying from
270 mV for
thioredoxin to
125 mV for DsbA have been attributed to the lowered
pKa values of their nucleophilic, active-site
cysteines and the difference in thermodynamic stability between their
oxidized and reduced forms (
Gox/red). The
lowered pKa of the nucleophilic cysteine thiols was
proposed to result from favorable interactions with the helix dipole
and charged residues in their vicinity. In this study, we have
eliminated all charged residues in the neighborhood of the active-site
disulfide of DsbA from Escherichia coli to analyze their
contribution to the physicochemical properties of the protein. We show
that the conserved charge network among residues Glu24,
Glu37, and Lys58 stabilizes the oxidized form
of DsbA and thus does not cause the high redox potential of the enzyme.
The pKa values of the nucleophilic cysteine
(Cys30) and the redox potentials of the DsbA variants E24Q,
E37Q, K58M, E24Q/K58M, E37Q/K58M, E24Q/E37Q, E24Q/E37Q/K58M, and
E24Q/E37Q/E38Q/K58M are similar to those of DsbA wild type. The redox
potentials of the variants neither correlate with the Cys30
pKa values nor with the
Gox/red values, demonstrating that the
relationship between these parameters is far more complex than
previously thought.
The formation of disulfide bridges is essential for folding and
stability of many secretory proteins. Numerous in vitro
studies have shown that disulfide bond formation is usually slow and
rate-limiting for folding (1-5), whereas proteins lacking disulfides
and cis X-proline peptide bonds may even fold within
milliseconds (6, 7). In vivo, disulfide bond formation is
catalyzed by disulfide oxidoreductases. All members of this class of
proteins possess an active-site disulfide bridge and the thioredoxin
fold (8, 9). The catalytic disulfide with the consensus sequence
CXXC (where X represents any amino acid) is
located at the amino terminus of an -helix. In eukaryotic
protein-disulfide isomerase
(PDI)1 and in the bacterial
enzymes DsbA, DsbC, and thioredoxin, only the more N-terminal of the
two active-site cysteines is solvent-exposed and acts as nucleophile in
disulfide exchange reactions (10-14).
The disulfide oxidoreductase DsbA is mainly responsible for the
efficient formation of disulfide bonds in proteins that are exported to
the periplasm of Escherichia coli (15, 16). The three-dimensional x-ray structure of oxidized DsbA was determined to
2.0-Å resolution and revealed that the enzyme consists of two domains,
the thioredoxin domain and an -helical domain of unknown function
(Fig. 1; Ref. 17). However, the structure of oxidized DsbA provides no
hints concerning its unusual physicochemical properties. These are
reflected by the extremely low pKa of the
nucleophilic active-site cysteine, Cys30, which has a value
of about 3.5 (18-20). The low pKa of
Cys30 qualitatively explains that reduced DsbA is more
stable than oxidized DsbA (21, 22), that it is the strongest oxidant in the family of disulfide oxidoreductases (21, 23), and that it undergoes
extremely fast reactions with thiol substrates (21, 24). Although the
residues of the XX dipeptide have been shown to influence
the pKa of DsbA Cys30 thiol (19), it is
still unclear whether charged residues in the vicinity of the
active-site helix or the helix dipole alone are responsible for the low
pKa of Cys30 in reduced wild type DsbA.
In the case of thioredoxin, values between 6.7 and ~9 have been
published for the pKa of the nucleophilic cysteine
Cys32 (10, 25-28). Besides charged residues in the
vicinity of the active-site helix, other explanations have been
proposed for a lowered pKa of the nucleophilic thiol
in human and E. coli thioredoxin such as the dipole of the
-helix (29-31), hydrogen bonding between the Cys32
thiolate and the amide of Cys35 (30, 32, 33), and a shared
proton between the two sulfurs of Cys32 and
Cys35 (26, 34).
Thioredoxin, PDI, and DsbA homologues possess a conserved amino acid
pair (Asp26/Lys57 in E. coli
thioredoxin, Glu47/Lys80 and
Glu391/Lys424 in human PDI, and
Glu24/Lys58 in E. coli DsbA (Fig.
2)) that is likely to form a buried salt bridge in the vicinity of the
active-site disulfide (17, 35-38). The hydrophobic environment of the
salt bridges gave rise to speculation that they may directly or
indirectly contribute to the lowered pKa values of
the nucleophilic cysteines by electrostatic interactions (38). In
addition, elevated pKa values of 7.5 and 9 were
determined for Asp26 of both oxidized and reduced
thioredoxin in the presence or absence of the salt bridge, respectively
(35, 36, 39-42), which was assumed to be directly linked to the redox
properties of thioredoxin. Fig. 2 shows the conserved charged residues
near the redox-active center of proteins with the thioredoxin fold.
Despite a low overall sequence homology, the charges of the side chains
corresponding to Glu24 and Lys58 of E. coli DsbA are invariant in the known DsbA sequences and are also
conserved in thioredoxins (with very few exceptions, e.g.
human thioredoxin, which lacks a corresponding lysine) and PDIs.
To study the influence of the Glu24/Lys58 salt
bridge on the physicochemical properties of DsbA from E. coli, we constructed the DsbA variants E24Q, K58M, and E24Q/K58M.
In contrast to thioredoxin, a second acidic residue, Glu37,
is located close to the Lys58 side chain in the structure
of E. coli DsbA (Fig. 1). An
acidic residue corresponding to Glu37 of E. coli
DsbA is present in 6 of the 10 known DsbA sequences (Fig.
2). In the x-ray structure of oxidized
E. coli DsbA, Glu37 is even closer to the
Lys58 side chain than Glu24 (the nearest
carboxylate oxygen of Glu37 is 3.4 Å apart from the
-amino group of Lys58 compared with 4.0 Å in the case
of Glu24). We recently characterized the physicochemical
properties of the DsbA variant E37Q, which are similar to those of DsbA
wild type (20). To further analyze the function of Glu37 in
the context of the neighboring residues Glu24 and
Lys58, we now investigated the properties of the double
variant E37Q/K58M, the triple variant E24Q/E37Q/K58M, and the quadruple
variant E24Q/E37Q/E38Q/K58M, which lacks all negatively charged
residues less than 13 Å apart from the sulfur of
Cys30.
Oligonucleotides were purchased from MWG-Biotech
(Ebersberg, Germany), and restriction enzymes were obtained from
Boehringer (Mannheim, Germany) or New England Biolabs (Schwalbach,
Germany). The plasmid pTHZ11 and E. coli THZ2 were kindly
provided by T. Zander (University Regensburg, Germany). Recombinant
hirudin was a gift from Hoechst AG (Frankfurt/Main, Germany). RNase A
was purchased from Boehringer or Fluka (Buchs, Switzerland). DE52 and
CM52 cellulose were from Whatman (Maidstone, United Kingdom), and PD-10
Sephadex columns were from Pharmacia (Freiburg, Germany). Tryptone and
yeast extract were from Difco, guanidinium chloride (GdmCl) was from
ICN (Meckenheim, Germany), and
isopropyl--D-thiogalactopyranoside (IPTG) was from AGS
(Heidelberg, Germany). 1,4-Dithio-DL-threitol (DTT),
reduced glutathione (GSH), oxidized glutathione (GSSG), dithionitrobenzoic acid,
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal),
and polymyxin B sulfate were purchased from Sigma-Aldrich (Deisenhofen,
Germany). All other chemicals were of analytical grade.
The plasmid pDSBA2 was
derived from plasmid pTHZ11 by deletion of the rop gene
after digestion with NspI and religation. pDSBA2 contains
the dsbA wild type gene under the control of the
trc promotor, the f1 and colE1
origins, the gene coding for ampicillin resistance, and the
lacIq gene. Site-directed mutagenesis was
performed with single-stranded, uridylated DNA from pDSBA2 according to
Kunkel et al. (43) using the Muta-Gene phagemid kit from
Bio-Rad (Glattbrugg, Switzerland). The following oligonucleotides were
used for mutagenesis (S corresponds to C or G): E24Q,
5-GAAGAAAGAGAAAAACTGCAGCACTTGCGGCGC-3
; E37Q/E38Q, 5
-CATTATCAGAAATATGCAGGACCTSTTSGAATTGATAGCAGTGCGGGCAG-3
; K58M, 5
-CCCATGAAGTTCACGTGATACATAGTCATCTTTACGCCTTCCG-3
. Mutations were identified by restriction analysis and verified by dideoxy sequencing using the Thermo SequenaseTM sequencing kit from Amersham
(Zürich, Switzerland).
Cells of
E. coli THZ2 (dsbA::kan,
recA::cam,
malF-lacZ102)2
were transformed with pDSBA2 harboring the respective dsbA
mutations. 4.5-liter cultures were grown in 2 × YT medium
supplemented with 100 mg/liter ampicillin and 50 mg/liter kanamycin at
30 °C. Production of DsbA was induced with 1 mM IPTG at
an optical density at 546 nm (A546) of 1.0, and
the cells were grown for another 6 h (final A546 = 3-3.5). The cells were harvested by
centrifugation and suspended in 150 ml of buffer (10 mM
MOPS/NaOH, pH 7.0, 5 mM EDTA, 150 mM NaCl, 1 mg/ml polymyxin B sulfate). After gentle stirring on ice for 2 h
and centrifugation (35,000 × g for 30 min), the supernatant (periplasmic extract) was dialyzed against 10 mM MOPS/NaOH, pH 7.0, or, in case of the variants E24Q/E37Q
and E24Q/E37Q/E38Q/K58M, against 10 mM Tris/HCl, pH 8.6. The extracts were applied to a DE52 cellulose column (40 ml). Proteins
were eluted with a linear gradient (500 ml) from 0 to 250 mM NaCl in the same buffers. Fractions containing DsbA were
pooled, dialyzed against 10 mM acetic acid/NaOH, pH 4.5, and applied to a CM52 cellulose column (40 ml). Proteins were eluted
with a linear gradient (500 ml) of 0-200 mM NaCl or 0-500
mM NaCl (in case of the variants E24Q/E37Q and
E24Q/E37Q/E38Q/K58M). Fractions containing DsbA were pooled, dialyzed
against distilled water, and stored at
20 °C. The yield of pure
DsbA was 10-15 mg/liter·A546. The molecular
mass of each DsbA variant was confirmed by electrospray mass
spectrometry (accuracy ±2). The amino acid replacements E24Q, E37Q,
and E38Q were also confirmed by N-terminal sequencing of residues
1-40. All DsbA variants were completely oxidized after purification,
since no free thiols were detectable by Ellman's assay (44). Protein
concentrations were determined by absorbance (A280, 1 mg/ml, 1 cm = 1.05).
dsbA
mutations were tested for complementation of E. coli THZ2
cells (dsbA) with respect to motility (45) and
formation of white colonies on agar plates containing X-gal (15, 19).
The ability to swarm was tested at 37 °C on 0.3% LB-agar plates
containing 100 mg/liter ampicillin and 50 mg/liter kanamycin.
-Galactosidase activity was tested at 37 °C on LB-agar plates
containing 0.4% maltose, 0.004% X-gal, 100 mg/liter ampicillin, and
50 mg/liter kanamycin.
Spectroscopic measurements were performed with a Hitachi F-4500 fluorescence spectrophotometer, a Varian Cary3E spectrophotometer, and a Jasco J-710 spectropolarimeter. A temperature of 25 °C was applied for all measurements, and buffer solutions were filtered before use (0.2-µm pore size). Far-UV and near-UV circular dichroism (CD) spectra of oxidized and reduced DsbA were recorded as described elsewhere (20, 23).
Unfolding/Refolding EquilibriaUnfolding/refolding experiments were essentially performed as described by Hennecke et al. (20). The exact GdmCl concentrations were determined from the refractive index of the solutions according to Nozaki (46). The original data of the transitions were analyzed according to the two-state model of folding using the six-parameter fit according to Santoro and Bolen (47).
Determination of Redox PotentialsFor determination of the equilibrium constants between DsbA and glutathione-oxidized DsbA (1 µM) was incubated with GSSG (100 µM) and different concentrations of GSH (0-900 µM) in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA under nitrogen atmosphere for 16 h at 25 °C. The equilibrium redox state of DsbA was measured by its fluorescence at 330 nm (excitation at 280 nm; Ref. 23). The equilibrium constant between DsbA and glutathione (Keq) was calculated by fitting the data according to Equation 1, where R is the fraction of reduced DsbA at equilibrium.
![]() |
(Eq. 1) |
The
pH-dependent ionization of the Cys30 thiol was
followed by the specific absorbance of the thiolate anion at 240 nm
(18, 48, 49). As a control, the pH-dependent absorbance of
the oxidized form of each DsbA variant was recorded. To avoid
precipitation artifacts and to minimize buffer absorbance, a buffer
system consisting of 10 mM K2HPO4,
10 mM boric acid, 10 mM sodium succinate, 1 mM EDTA, and 200 mM KCl (containing 100 µM -mercaptoethanol for the reduced proteins) was
used. The pH (initial value, 8.5) was lowered to 2.2 by stepwise
addition of aliquots of 0.1 M HCl, and the absorbance at
240 and 280 nm was recorded and corrected for the volume increase.
Samples had an average initial protein concentration of 25 µM. The pH dependence of the thiolate-specific absorbance
signal (S = (A240/A280)reduced/(A240/A280)oxidized) was fitted according to the Henderson-Hasselbalch equation,
![]() |
(Eq. 2) |
Reduced, unfolded
RNase A and hirudin (A280, 1 mg/ml, 1 cm = 0.56 and 0.37, respectively) were prepared as described previously
(24). The quantitative reduction of the proteins was verified by
Ellman's assay (44). Oxidation of RNase A and hirudin by DsbA was
analyzed fluorometrically at 25 °C with an SX-17MV stopped-flow
reaction analyzer (Applied Photophysics) using an excitation wavelength
of 295 nm and emission wavelengths 320 nm. Pseudo-first-order
conditions were applied using 1 µM oxidized DsbA and 200 µM free thiols of the respective substrate protein or
DTT. Buffer conditions were 100 mM formic acid/NaOH, pH
3.0, 100 mM formic acid/NaOH, pH 4.0, 100 mM
acetic acid/NaOH pH 5.0, 100 mM MES/NaOH, pH 6.0, 100 mM sodium phosphate, pH 7.0, 100 mM Tris/HCl,
pH 8.0, 100 mM Bicine/NaOH, pH 9.0, and 100 mM glycine/HCl, pH 10.0. All buffers contained 1 mM EDTA and
were degassed and flushed with nitrogen before use. Polypeptide
specificity is defined as the ratio between the apparent second-order
rate constant of the oxidation of the respective substrate protein and
the apparent second-order rate constant of the oxidation of DTT.
We constructed the plasmid pDSBA2, which contains the dsbA wild type gene under control of the IPTG-inducible trc promotor, the lacIq gene, and the phage f1 origin (see "Experimental Procedures"). Thus, the plasmid combines features necessary for in vivo complementation, efficient overexpression of dsbA, and production of single-stranded DNA for site-directed mutagenesis (43). The following DsbA variants were generated: E24Q, K58M, E24Q/K58M, E24Q/E37Q, E37Q/K58M, E24Q/E37Q/K58M, and E24Q/E37Q/E38Q/K58M. All variants could be overproduced in the dsbA-deficient E. coli strain THZ2 (see below) to approximately the same extent as the wild type protein. They were purified to homogeneity by conventional chromatographic techniques in the absence of reducing agents and were all obtained in the oxidized form with yields of about 50 mg/liter bacterial culture.
Complementation of the dsbA-deficient E. coli Strain THZ2We
used the E. coli strain THZ2 to characterize the in
vivo function of the DsbA variants. This strain has a
Tn10::kan insertion in the chromosomal
dsbA gene and possesses a gene coding for a MalF--galactosidase fusion protein (15, 19, 50). The following criteria were used to determine the ability of the DsbA variants to
complement the dsbA
phenotype of THZ2 after
transformation with the respective dsbA expression plasmids: (i)
restoration of motility on agar plates containing 0.3% agar, since
THZ2 is immotile due to the lack of disulfide bonds in the P-ring
protein of the flagellar motor (45), and (ii) generation of a
Lac
phenotype due to oxidative inactivation of
periplasmic
-galactosidase by functional DsbA (15, 19), which
results in white colonies on agar plates containing X-gal. Even in the
absence of IPTG, all DsbA variants yielded a DsbA+
phenotype of transformed THZ2 cells, which was indistinguishable from
the phenotype of cells harboring the expression plasmid for wild type
DsbA.
Like for DsbA wild type (23), a 3-fold increase in tryptophan fluorescence was observed for the variants E24Q, K58M, E24Q/K58M, and E37Q/K58M when the active-site disulfide bond was reduced. However, all variants in which two or three negative charges had been exchanged (E24Q/E37Q, E24Q/E37Q/K58M, and E24Q/E37Q/E38Q/K58M) exhibited an approximately 2-fold higher fluorescence of the oxidized form compared with oxidized wild type. Therefore, their fluorescence increased only about 1.5-fold upon reduction. This difference was still sufficient to determine their thermodynamic stabilities and redox equilibria with glutathione by fluorescence spectroscopy (see below). The emission maxima of all oxidized and reduced variants were identical to those of wild type DsbA (data not shown).
Far-UV and near-UV CD spectra were recorded to detect possible conformational changes in the DsbA variants. The spectra of the oxidized and reduced variants were identical to those of oxidized and reduced wild type (data not shown). Thus, the native tertiary structure of oxidized and reduced DsbA was essentially not affected in any of the variants.
Thermodynamic Stabilities of the Oxidized and Reduced DsbA VariantsThe free energies of stabilization of the oxidized and
reduced forms of the variants were measured fluorometrically by
GdmCl-induced equilibrium transitions assuming a two-state model of
folding (Fig. 3; Table
I). All transitions were fully
reversible, with cooperativities of 20-27 kJ/mol·M for
the oxidized variants and 22-27 kJ/mol·M for the reduced
variants. Only the reduced variant E24Q/E37Q/E38Q/K58M exhibited a
strongly reduced cooperativity of 15 kJ/mol and thus may not fold
according to the two-state model (51).
|
A unique property of the DsbA wild type protein is its destabilized oxidized form (21, 22). In all variants investigated, the oxidized form was also less stable than the reduced form. Removal of the negative charge of residue Glu24 in the variant E24Q slightly stabilized both the reduced and oxidized forms about 2 kJ/mol, whereas the exchanges E37Q and K58M strongly destabilized the oxidized forms of the variants 8-9 kJ/mol and stabilized the reduced forms about 2 kJ/mol. The stabilities of the oxidized and reduced double variants E24Q/K58M, E37Q/K58M, and E24Q/E37Q revealed that the stability changes caused by the single variants were not additive. Analysis of the stabilities according to the concept of double mutant cycles (Ref. 52; Table II) shows that each of the interactions Glu24-Lys58, Glu37-Lys58, and Glu24-Glu37 energetically stabilizes the oxidized form of DsbA wild type and should principally counteract its oxidative force. Qualitatively, similar contributions of the interactions Glu24-Lys58, Glu37-Lys58, and Glu24-Glu37 to the stability of oxidized and reduced DsbA are observed on the background of the E37Q, E24Q, and K58M variants, respectively. The stabilities of the oxidized and reduced triple variant E24Q/E37Q/K58M also show the whole interaction network between the residues Glu24, Glu37, and Lys58, which stabilizes the oxidized form of the wild type.
|
Overall, the measured stability differences between the oxidized and
reduced variants (Gox/red) predicted more
oxidizing proteins in the case of the variants K58M, E37Q, E37Q/K58M,
and E24Q/E37Q/K58M and more reducing proteins in the case of the
variants E24Q/K58M and E24Q/E37Q (Table I).
The redox potentials
(E0) of the DsbA variants were
measured at pH 7.0 and 25 °C by determining their equilibrium constants (Keq) with glutathione using a value
of
240 mV for the standard potential of the GSH/GSSG redox couple
(53). The equilibrium constants were measured fluorometrically,
assuming no significant equilibrium concentrations of DsbA-glutathione mixed disulfides (23), and yielded reliable data without systematic deviations from theory (Fig. 4; Table
III). Surprisingly, the redox potential
of none of the variants differed more than 10 mV from that of the DsbA
wild type, although the stability differences between their oxidized
and reduced forms (
Gox/red) predicted that
the redox potentials should be 26-77 mV more oxidizing in the case of
the variants K58M, E37Q, E37Q/K58M, and E24/E37Q/K58M and 10-14 mV
more reducing in the case of E24/K58M and E24Q/E37Q (Table III). A
qualitative agreement between the measured and the predicted changes in
redox potential was only obtained for the variants K58M and E24Q/K58M.
The lack of correlation between the measured values of
Gox/red and Keq is
best shown by the variant E37Q/K58M, which is 8 mV more reducing than
DsbA wild type and predicted to be 77 mV more oxidizing.
|
To test whether the
measured differences between the redox potentials of the variants and
DsbA wild type were caused by changes in the pKa
value of the nucleophilic Cys30 thiols, we determined the
pH dependence of the fraction of the Cys30 thiolate in all
reduced variants by their specific absorbance at 240 nm, using oxidized
DsbA as a reference (Fig. 5, Table III) (18-20, 48, 49). A pKa of 3.55 was measured for
wild type DsbA, which is identical within experimental error with
previously published data (18-20). The Cys30
pKa values of the variants varied between 3.19 (K58M) and 4.46 (E24Q/K58M), which again demonstrated that direct or indirect electrostatic interactions between the Cys30
thiolate and the charged residues in the vicinity of the active-site helix do not significantly affect the reactivity of the nucleophilic cysteine. Due to the general dependence of disulfide exchange equilibria on the pKa values of the involved thiols
(54) and assuming that the amino acid replacements only affected the pKa value of the Cys30 thiol but not
that of the Cys33 thiol, we expected decreased
pKa values of Cys30 in the more
oxidizing variants and increased pKa values in the
more reducing variants. However, there was no general correlation between the Cys30 pKa values and the
measured redox potentials (Table III). For example, the most oxidizing
variant E24Q/E37Q even showed an increased pKa of
the Cys30 thiol.
pH Dependence of the Polypeptide Specificity of the DsbA Variants
It was shown previously that DsbA possesses a peptide
binding site (17). This explains its specificity toward unfolded
polypeptide substrates that are randomly oxidized by the enzyme (20,
21, 24, 55-57). DsbA's polypeptide specificity is higher at acidic pH
and independent of the isoelectric point of the polypeptide substrate
(20). To test whether the charge network
Glu24-Glu37-Lys58 affects the
polypeptide specificity of DsbA, we measured the reactivities of the
oxidized variants toward reduced, unfolded RNase A (pI = 9.6; Ref.
58) and hirudin (pI = 3.9; Ref. 59) between pH 3 and pH 10 and
compared them with their reactivities toward DTT. Since both hirudin
and RNase A lack tryptophan residues, the reactions were followed by
the increase in DsbA's tryptophan fluorescence in a stopped-flow
fluorescence spectrometer. The polypeptide specificity of DsbA was
defined as the ratio between the apparent second-order rate constant of
random oxidation of a reduced substrate protein and the apparent
second-order rate constant of oxidation of DTT
(kprotein/kDTT). Fig.
6 shows the pH dependence of the
polypeptide specificities of all variants for the substrates RNase A
and hirudin. Like in DsbA wild type, the peptide specificity of
variants is most pronounced at acidic pH and disappears at pH values
above 9. Overall, the values of kprotein/kDTT do not
differ more than a factor of 4 from those of wild type DsbA. The
individual values of kprotein and
kDTT were also very similar to those of wild
type DsbA (data not shown). Interestingly, the peptide specificity of
the variant E24Q is present over a broader pH range compared with the
wild type and all other variants and is still significantly pronounced
between pH 7 and pH 8 (Fig. 6). The only variant that appears to be
sensitive toward the charge (pI) of the polypeptide substrate proved to be the variant E24Q/K58M, which exhibits the highest
kprotein/kDTT values for
RNase A and the lowest
kprotein/kDTT values for
hirudin at acidic pH (Fig. 6).
The nucleophilic active-site cysteine thiols of Cys30
in DsbA and Cys32 in thioredoxin have very low
pKa values of approximately 3.5 (18-20) and 7.5 (27, 60), respectively, compared with 9.5 for a normal cysteine (61).
This difference has been proposed to be the main determinant of the
redox properties of these enzymes (18). The thiolate anion that is
located at the N terminus of an -helix has been suggested to be
stabilized by the partial positive charge of the helix dipole and by
additional electrostatic interactions between charged residues and the
active-site disulfide (18, 27, 31, 38, 62). In this study we focused on
the interaction network of all charged residues (Glu24,
Glu37, Glu38, and Lys58) in the
vicinity of the active site of DsbA because at least Glu24
and Glu37 may form an ion pair with Lys58 in
the structure of oxidized DsbA (17).
The residues corresponding to Glu24 and Lys58 in DsbA, e.g. Asp26 and Lys57 in E. coli thioredoxin, are in general conserved in disulfide oxidoreductases (Fig. 2). In thioredoxin, an acidic residue corresponding to Glu37 or Glu38 in DsbA is absent, and thus a buried salt bridge is very likely to be formed solely between Asp26 and Lys57. Importantly, an increased pKa value of Asp26 was observed, which was attributed to the hydrophobic environment of this residue (27, 35, 36, 39-42). An abnormal pKa of an acidic residue in a native protein influences protein stability pH-dependently as shown by the Equation 3 (35, 36),
![]() |
(Eq. 3) |
Accordingly, the oxidized variant D26A was demonstrated to be 15.5 kJ/mol more stable than the wild type at pH 7.0 (35, 36). On the other
hand, the pKa of Asp26 does not explain
why oxidized thioredoxin is thermodynamically favored over the reduced
form by 10 kJ/mol at pH 7.0 (63), since the pKa was
demonstrated to be invariant with a value of 7.5 in both oxidized and
reduced thioredoxin (40). Removal or absence of the salt bridge
Asp26/Lys57 in the variant K57M or in human
thioredoxin (which lacks the residue corresponding to
Lys57), respectively, results in an increased
pKa of 9.4 for Asp26 (41, 42) and should
therefore destabilize the protein (35, 36). Nevertheless, assuming a
pKa (u) of 3.9 (61), a
G of only +0.7 kJ/mol at pH 7.0 (Equation 3) is
predicted, and consequently, the pKa of
Asp26 cannot account for the stability difference between
the oxidized and reduced form of thioredoxin at physiological pH.
Interestingly, the exchange K57G had no effect on the
pKa of Asp26 in oxidized thioredoxin
(27).
The same considerations principally apply to the buried side chain of
Glu24 in DsbA and the putative salt bridge with
Lys58. Glu24 was proposed to be the residue
titrating at pH 6.7 and increasing the reactivity of the
Cys30 thiolate with iodoacetamide about 4-fold (18).
Assuming a pKa (f) of 6.7 (18) and a
pKa (u) of 4.4 (61), the removal of the
negative charge is predicted to stabilize the variant E24Q 12.1 kJ/mol
at pH 7.0 (Equation 3). However, we found only a slight stabilization
of 2.2 kJ/mol for reduced and 2.3 kJ/mol for oxidized E24Q (Table I).
If the pKa of Glu24, as is the case for
Asp26 of thioredoxin, increased about 2 units in the
variant K58M, a G of +2.7 kJ/mol at pH 7.0 should
be expected. Instead, values of +8.3 and
3.0 kJ/mol were observed for
the oxidized and reduced variant, respectively (Table I), which cannot
be explained by an altered pKa of Glu24.
The same considerations apply for Glu37, which could also
form a salt bridge with Lys58 and which is an alanine in
thioredoxin (Fig. 2).
In contrast to the D26A exchange in thioredoxin, which caused an increase of the Cys32 pKa of 0.4-0.9 units (27, 41), the pKa of Cys30 in the DsbA variant E24Q was unchanged (Table III). The function of Glu24 in DsbA therefore appears to be different from that of Asp26 in thioredoxin. This might result from the presence of Glu37, but the double variant E24Q/E37Q is destabilized 5.4 and 8.0 kJ/mol for the oxidized and reduced forms, respectively, although the Cys30 pKa is increased 0.3 units (Tables I and III).
It can be concluded that, in agreement with the data available for thioredoxin, the pKa values of Glu24 and Glu37 most likely do not contribute to the stability difference between the oxidized and reduced form of DsbA either in the presence or in the absence of the buried salt bridge with Lys58. Nevertheless, altered stabilities of both forms can be detected with the DsbA variants. Whether Glu24 indeed modulates the reactivity of Cys30 in DsbA still has to be tested by the pH dependence of the reactivity of Cys30 in the E24Q variant.
The analysis of the thermodynamic stabilities of the variants with the concept of double mutant cycles (52) reveals another unexpected feature of the supposed ion pairs Glu24-Lys58 and Glu37-Lys58 because they appear to counteract the oxidative force of wild type DsbA by thermodynamically stabilizing the oxidized form. The same is valid for the interaction between Glu24 and Glu37 (Table II).
Correlation of Stabilities, pKa Values of the Cys30 Thiol, and Redox PotentialsIn principle, the
following predictions can be made for the relationship between the
difference of the thermodynamic stabilities of the oxidized and reduced
form (Gox/red), the pKa of the Cys30 thiol, and the equilibrium constant with
glutathione (redox potential) of DsbA. (i) The
Gox/red at a given pH should be
predictable if it is assumed that the oxidation of DsbA causes nothing
but the removal of the negative charge of Cys30 (18). (ii)
Equilibrium constants of disulfide exchange reactions can be predicted
if the pKa values of all thiols involved are known
(54). Consequently, a change in the pKa of Cys30 in a DsbA variant should alter the redox potential
(
E0
WT) (18,
19). (iii)
Gox/red should also directly correlate with
E0
WT. Although
a correlation between changes in the pKa of
Cys30 and the redox potentials could recently be
demonstrated for a series of DsbA variants in which the XX
dipeptide between the active-site cysteines had been randomly mutated
(19), Table III shows that the above assumptions are not consistent
with the results obtained for DsbA variants devoid of charges in the
vicinity of the active site. The situation seems to be far more
complex. The most extreme example is the variant E37Q/K58M, which is 8 mV more reducing than the wild type, but the
Gox/red value predicts an increase of the
redox potential of 77 mV (Table III). Overall, the measured redox
potentials of the DsbA variants correlate neither with the values of
Gox/red nor with the Cys30
pKa values. What are the possible explanations for
these deviations from theory? First, the assumption that the
pKa of the buried active-site cysteine
Cys33 is normal and not affected by residue replacement
(18) may not be justified. Since the absorbance of DsbA at 240 nm
changes strongly at alkaline pH, we were not able to measure the
pKa of Cys33 accurately by the
difference in A240 between the oxidized and reduced variants. Single exchanges in the charge network
Glu24-Glu37-Glu38-Lys58
of DsbA are likely to change the pKa values of the
other residues in the network, i.e. the loss of a negative
charge at one position may be compensated by a decreased
pKa of another acidic residue. Second, at least some
of the exchanged, charged residues may only affect the stabilities of
the oxidized and reduced forms of the protein but not the
pKa values of the active-site cysteines. In this
context, the properties of the DsbA variant K58M are remarkable.
Although the removal of the positive charge of Lys58 is
expected to cause an accumulation of negative charges in the vicinity
of the active site, the pKa of Cys30 is
even further decreased to 3.2 (Table III), which is, to our knowledge,
the lowest pKa of a cysteine thiol in a native protein known so far. Therefore, we believe that especially the pH
dependence of
Gox/red and the equilibrium
constants with glutathione will have to be measured for DsbA wild type
and some of the variants to gain further insights into the relationship between
Gox/red,
E0
, and the
pKa of the Cys30 thiol.
It is obvious that neither charge of the residues Glu24, Glu37, Glu38, and Lys58 is required for efficient recycling of oxidized DsbA by DsbB in vivo, since all variants restored the DsbA+ phenotype of the dsbA-deficient E. coli strain THZ2. The broad polypeptide specificity of DsbA was found to be most pronounced at acidic pH but was largely unaffected in the variants. Only the variants E24Q and E24Q/K58M showed higher polypeptide specificity also between pH 7 and 8 and a slightly increased preference for RNase A compared with hirudin, respectively (Fig. 6). The x-ray structure of oxidized DsbA supports the view that charges near the active site are not critical for peptide recognition (17). DsbA has a conserved groove within the thioredoxin domain, which is located next to the active-site disulfide and composed of hydrophobic and uncharged polar residues.
Conservation of the Buried Salt BridgeThe results presented in this paper raise the question of why the residues corresponding to Glu24 and Lys58 in DsbA are so well conserved within the disulfide oxidoreductase family. The reactivity of oxidized DsbA toward reduced polypeptides and DTT and the other redox properties investigated are practically independent of the presence of Glu24 and Lys58 or even of the interaction between Glu24 and Glu37. One possible explanation could be that the Glu24-Lys58 ion pair has a distinct function in the folding pathway of thioredoxin-like proteins. In the case of barnase, a buried salt bridge has been shown to direct the folding pathway without stabilizing the native structure (64, 65). Thus, as in barnase, ionic interactions could play an important role in the early condensation of the disordered polypeptide chain of disulfide oxidoreductases by restricting the number of possible conformations of early folding intermediates, which therefore may result in the acceleration of folding.
We thank T. Zander and J. C. A. Bardwell for providing the plasmid pTHZ11 and the strain THZ2, G. Frank for N-terminal sequencing, P. James for recording electrospray mass spectra, and V. Eggli for construction of the E24Q variant.