Influence of Acidic Residues and the Kink in the Active-site Helix on the Properties of the Disulfide Oxidoreductase DsbA*

(Received for publication, August 7, 1996, and in revised form, October 7, 1996)

Jens Hennecke , Chantal Spleiss and Rudi Glockshuber Dagger

From the Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The catalytic disulfide bond Cys30-Cys33 of the disulfide oxidoreductase DsbA from Escherichia coli is located at the amino terminus of an alpha -helix, which has a kink caused by insertion of a tripeptide (residues 38-40). The oxidative force of DsbA (E'0 = -125 mV) mainly results from the low pKa of 3.4 of its Cys30 thiol. To investigate the role of the kink and the electrostatic contribution of Glu37 and Glu38 to the redox properties of DsbA, we have characterized a series of DsbA variants (Delta 38-40, Delta 38-40/H41P, E37Q, E38Q, and E37Q/E38Q). In contrast to theoretical predictions, the redox potentials of the variants are almost unchanged, and the pKa values of Cys30 do not differ by more than 0.5 units from that of DsbA wild type. All variants show the same in vivo activity and dependence of redox potential on ionic strength as the wild type. The mutations have no influence on the polypeptide specificity of the protein, which is independent of the isoelectric point of the polypeptide substrate and most pronounced at acidic pH. We conclude that neither the kink in the active-site helix nor Glu37 and Glu38 are critical for the physical properties of DsbA.


INTRODUCTION

DsbA is a soluble, monomeric 21.1-kDa protein (189 amino acids) that is required for efficient disulfide bond formation in the periplasm of Escherichia coli (1, 2). The enzyme contains a single, catalytic disulfide with the active-site sequence Cys30-Pro31-His32-Cys33. DsbA-catalyzed disulfide bond formation in the periplasm of E. coli involves two steps. First, the disulfide form of DsbA oxidizes reduced, folding substrate proteins. In the second step, DsbA becomes reoxidized by a disulfide exchange reaction with DsbB, a protein located in the inner E. coli membrane (3, 4, 5, 6).

The three-dimensional x-ray structure of oxidized DsbA (7) has revealed that the enzyme possesses the thioredoxin fold, which is common to all known structures of disulfide oxidoreductases (8). In addition to its thioredoxin domain, DsbA possesses a second domain of 76 residues, which is inserted into the thioredoxin motif and exclusively consists of alpha -helices (7) (Fig. 1A).


Fig. 1. Comparison of the active-site helices in DsbA, glutaredoxin, and thioredoxin. A, Molscript diagram (9) of the x-ray structure of DsbA from E. coli (7). The thioredoxin-like domain of DsbA, with the kinked active-site helix indicated in black, is shown at the bottom, and its alpha -helical domain is shown at the top. The cysteine side chains and residues mutated in this study are also indicated. Structural (B) and sequential (C) alignment of the active-site helices of DsbA, E. coli thioredoxin (10), and E. coli glutaredoxin (11) is also shown. The tripeptide insertion Glu38-Val39-Leu40 is responsible for the kink in DsbA, and a proline residue causes the kink in thioredoxins, whereas the active-site helix of glutaredoxin is straight.
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The biophysical characterization of DsbA has shown that the enzyme is the strongest known oxidant within the disulfide oxidoreductase family (12, 13) and oxidizes thiol compounds extremely rapidly (13, 14, 15, 16). In addition, DsbA specifically reacts with reduced, unfolded polypeptides, which can be explained by the presence of a polypeptide binding site (14, 15, 16, 17). DsbA is thus ideally suited as final oxidant of protein disulfides in the periplasm. The physical properties of DsbA are linked to the pKa of the nucleophilic thiol of Cys30, which is extremely low and has a value of 3.5 compared with pKa values of about 9 for normal cysteine side chains (18). On the one hand, the low pKa of Cys30 can explain the fact that the oxidized form of DsbA is about 20 kJ/mol less stable than its reduced form at neutral pH (13, 19) when one assumes that the oxidation of DsbA is nothing but the removal of the negative charge on the Cys30 thiol (18). On the other hand, the low pKa explains the high rate constants of disulfide exchange reactions involving DsbA (18, 20). The interdependence of the redox potential, the stability difference, and the pKa value of Cys30 has recently also been demonstrated by Grauschopf et al. (21) for a series of DsbA variants with amino acid replacements in the dipeptide sequence between the active-site cysteines.

In the known structures of disulfide oxidoreductases, the active-site disulfide is located at the amino terminus of an alpha -helix (8). Interestingly, no positively charged residue is located in the vicinity of the Cys30 side chain in the structure of DsbA that could explain its low pKa. It was therefore postulated that the helix dipole as well as the amino acid composition of the active-site helix may be responsible for stabilizing the negative charge on Cys30 (18, 22). In addition, electrostatic calculations suggested that the side chains of Glu37 and Glu38 as well as the backbone conformation of residues 38-40 have an influence on the redox properties and the low pKa value of the Cys30 thiol (23, 24). Comparison of the amino acid sequences and three-dimensional structures of the active-site helices of DsbA, thioredoxin, and glutaredoxin shows that the active-site helix of glutaredoxin is straight, whereas the corresponding helices in DsbA and thioredoxin are kinked (Fig. 1, B and C). In DsbA, the kink is about 60° and appears to be caused by the insertion of the tripeptide Glu38-Val39-Leu40. The active-site helix of thioredoxin has a kink of about 30°, which is caused by a conserved proline residue at the corresponding position.

In this study, we address the question of whether the residues that cause the kink in the active-site helix of DsbA are responsible for the unusual biophysical properties of the enzyme. For this purpose, we have constructed a DsbA variant in which the tripeptide insertion was deleted (Delta EVL), as well as a double variant, where His41 was additionally replaced by proline (Delta EVL/H41P) to mimic the active-site helices of glutaredoxin and thioredoxin, respectively. Since the kink in the active-site helix may also determine the orientation of the catalytic disulfide relative to the peptide binding site, we have investigated the influence of the mutations on the polypeptide specificity of DsbA. Since Glu37 and Glu38 may contribute electrostatically to the pKa of Cys30 and are part of an unusual acidic patch on the surface of DsbA (7), we replaced these residues by glutamines in another set of variants.


EXPERIMENTAL PROCEDURES

Materials

1,4-Dithio-DL-threitol (DTT),1 reduced glutathione (GSH), oxidized glutathione (GSSG), sodium succinate, amino acids, ampicillin, dithionitrobenzoic acid, 5-bromo-4-chloro-3-indoyl beta -D-galactoside, and polymyxin B sulfate were purchased from Sigma (Deisenhofen, Germany). Guanidinium chloride (GdmCl) and maltose were from ICN, isopropyl-beta -D-thiogalactoside (IPTG) was from AGS GmbH (Heidelberg, Germany), DE52- and CM52-cellulose were purchased from Whatman (Maidstone, United Kingdom), and phenyl-Sepharose was obtained from Pharmacia (Uppsala, Sweden). All other chemicals were from Merck (Darmstadt, Germany) and of highest purity available. The strain THZ2 was kindly provided by T. Zander (University of Regensburg, Germany).

Site-directed Mutagenesis

For construction and expression of the dsbA mutants, the phasmid pDsbA2 was used, where the dsbA gene is under control of the trc promotor and its periplasmic expression is inducible by IPTG. pDsbA2 was derived from the previously described expression plasmid pJW1717 (21) after deletion of the rop gene.2 Single-stranded DNA was obtained after transformation of E. coli CJ236 and infection with the helper phage M13KO7 (25). Site-directed mutagenesis was performed according to Kunkel et al. (26, 27) using the kit supplied by Bio-Rad. The following oligonucleotides were used (S corresponds to C or G): E37Q and E38Q, 5'-C ATT ATC AGA AAT ATG CAG GAC CTS TTS GAA TTG ATA GCA GTG CGG GCA G-3'; Delta EVL, 5'-CAC ATT ATC AGA AAT GTG CTC AAA CTG ATA GCA G-3'; Delta EVL/H41P, 5'-CTT CAC ATT ATC AGA AAT AGG CTC AAA CTG ATA GCA G-3'.

Mutants were identified by restriction analysis and verified by complete sequencing of the mutated genes using the T7 sequencing kit from Pharmacia.

Expression and Purification of DsbA

Cells of E. coli THZ2 (dsbA::kan, recA::cam, lambda malF-lacZ102; with respect to this genotype, THZ2 is isogenic to THZ7; Ref. 21) harboring the respective expression plasmids were grown in 2 × YT medium containing 100 mg/liter ampicillin at 37 °C. After induction with 1 mM IPTG at an optical density of 1.0 (546 nm), the cells were grown for 12 h at room temperature. The cells were harvested by centrifugation, suspended in ice-cold extraction buffer (10 mM MOPS/NaOH, pH 7.0, 5 mM EDTA, 150 mM sodium chloride, 1 mg/ml polymyxin-B-sulfate; 2 ml/g wet cells), stirred for 1 h at 4 °C, and centrifuged at 35,000 × g for 30 min. The supernatant (periplasmic extract) was dialyzed against buffer A (10 mM MOPS/NaOH, pH 7.0) and applied to a DE52-cellulose column (15 ml). The protein was eluted with a linear gradient (500 ml) from 0 to 500 mM sodium chloride in buffer A. Fractions containing DsbA were pooled, adjusted to 1.2 M ammonium sulfate and pH 8.0 by the addition of 4 M ammonium sulfate and 1 M Tris, respectively, and applied to a phenyl-Sepharose column (15 ml). The column was washed stepwise with 1.2 and 0.8 M ammonium sulfate in buffer B (20 mM Tris/HCl, pH 8.0, 100 mM sodium chloride). The protein was eluted by a linear gradient (125 ml) from 0.8 M ammonium sulfate in buffer B to 20 mM Tris/HCl, pH 8.0. Fractions containing DsbA were dialyzed against 10 mM acetic acid/NaOH, pH 4.5 (buffer C). The protein was applied to a CM52 column (15 ml) and eluted by a linear gradient (500 ml) from 0 to 500 mM sodium chloride in buffer C. Fractions containing pure DsbA were dialyzed against distilled water and stored at -20 °C.

The molecular mass of each variant was confirmed by electrospray mass spectroscopy (error <= 1 Da). The correct amino termini of the variants E37Q, E38Q, and E37Q/E38Q were also confirmed by Edman sequencing of residues 1-45.

Protein concentrations were determined by measuring the absorbance at 280 nm (native, oxidized DsbA: epsilon 280 = 23,300 M-1 cm-1) (28). All purified DsbA variants were obtained in the oxidized form, as shown by the lack of free thiol groups (29).

Determination of DsbA Activity in Vivo

The dsbA mutants were tested for their ability to complement the phenotypes of E. coli dsbA- strains, namely the lack of motility (30) and the ability to express functional beta -galactosidase in the periplasm (1). For all assays, E. coli THZ2 (see above; Ref. 21) was used. Motility assays were performed at 37 °C on 0.3% agar plates containing ampicillin (100 mg/liter) and either rich medium (2 × YT) or minimal medium supplemented with all amino acids except cysteine and cystine (31). beta -Galactosidase activity was tested at 37 °C on indicator plates containing one of the above media, supplemented with 0.4% maltose, 0.004% 5-bromo-4-chloro-3-indoyl beta -D-galactoside, 1.5% agar, and 100 mg/liter ampicillin.

Circular Dichroism Measurements

Far- and near-UV CD spectra were measured at 25 °C on a Jasco 710 CD spectropolarimeter in 0.02- and 1-cm quartz cuvettes, respectively. Protein concentrations of 35 µM in 1 mM sodium phosphate, 10 mM sodium sulfate, pH 7.0, were used. Samples with reduced DsbA contained 100 µM DTT.

GdmCl-induced Unfolding Equilibria

For unfolding, proteins (50 µM in distilled water) were diluted 1:50 with degassed 100 mM sodium phosphate, pH 7.0, 1 mM EDTA containing different concentrations of GdmCl and incubated at 25 °C for 3 days. In the case of reduced DsbA, DTT (1 mM) was included. For refolding experiments, DsbA was denatured at concentrations of 50 µM in 4 M GdmCl, 100 mM sodium phosphate, pH 7.0, 1 mM EDTA (additionally containing 50 mM DTT in the case of reduced DsbA) for 1 h at room temperature prior to the 1:50 dilution. The transitions were followed by the fluorescence at 365 nm and at 327 nm for oxidized and reduced DsbA, respectively (excitation at 280 nm), using a Hitachi F-4000 spectrofluorimeter. Data were evaluated according to a two-state equilibrium with a six-parameter fit as described previously (19, 32).

Determination of the Redox Potential

The redox potentials were determined fluorimetrically by measuring the equilibrium constant between DsbA and glutathione, assuming no significant equilibrium concentrations of DsbA/glutathione mixed disulfides (12). DsbA (1 µM) was incubated under a nitrogen atmosphere with 0.1 mM GSSG and different concentrations of GSH (0.003-3 mM) in 100 mM sodium phosphate, pH 7, 1 mM EDTA for 20 h at 25 °C. The relative amount of reduced DsbA at equilibrium (R) was measured using the specific DsbA fluorescence at 332 nm (excitation at 280 nm). The equilibrium constant (Keq) was determined by fitting the data to the equation, R = ([GSH]2/[GSSG])/(Keq + ([GSH]2/[GSSG])). The redox potential was calculated using the Nernst equation and a value of -240 mV as the standard redox potential of glutathione (33).

Determination of pKa Values

The pKa values of the thiol group of Cys30 were measured by the increase in absorbance at 240 nm upon formation of the thiolate (18). Oxidized DsbA was used as a reference. For preparation of reduced DsbA, the protein (0.15 mM in distilled water) was reduced by incubation with DTT (0.5 mM) for several minutes. DTT was removed by gel filtration on a PD 10 column (Pharmacia) equilibrated with 10 mM K2HPO4, 10 mM boric acid, 10 mM sodium succinate, 1 mM EDTA, and 200 mM potassium chloride (pH 8). The reference (oxidized DsbA) was subjected to the same gel filtration procedure.

The pH titration was performed with an initial DsbA concentration of 20 µM and an initial volume of 2 ml. The pH was lowered by the stepwise addition of 20-50 µl of 0.1 M HCl, and A240 and A280 were recorded for 10 s, averaged, and corrected for the volume increase. Since A280 proved to be essentially independent of pH the value of (A240, red/A280, red)/(A240, ox/A280, ox) was used to measure the fraction of the thiolate anion. Data were fitted according to the Henderson-Hasselbalch equation.

Determination of Peptide Specificity

As a measure of the peptide specificity of DsbA, we used the factor by which it oxidizes reduced hirudin and RNaseA faster than the small dithiol DTT. Reduced, unfolded RNaseA and hirudin were prepared essentially as described by Wunderlich et al. (14) and Otto and Seckler (34), except that gel filtration was performed in 1 mM HCl, 200 mM KCl. Oxidized DsbA (1 µM) was reacted with reduced, unfolded RNaseA, hirudin, or DTT (100 µM total concentration of free thiols, pseudo-first-order conditions) at 25 °C in 20 mM K2HPO4, 20 mM boric acid, 20 mM sodium succinate, 200 mM potassium chloride, 1 mM EDTA (adjusted with HCl to pH 4-10).

The reaction was followed by the increase in DsbA fluorescence (lambda ex = 295 nm; lambda em >=  305 nm) on a SX-17MV stopped-flow reaction analyzer (Applied Photophysics, Leatherhead, UK). In principle, the fluorescence increase of DsbA only detects the formation of the DsbA/substrate mixed disulfide, since previous investigations had shown that the mixed disulfide between glutathione and DsbA (at Cys30) exhibits the same fluorescence characteristics as reduced DsbA.3 However, the intramolecular attack of the mixed disulfide is very rapid. Therefore, the measured apparent rate constants should be identical with the overall rate constant of DsbA reduction. As reported previously (14), the fluorescence increase upon reduction with DTT was fully consistent with a second-order reaction. This was verified by the linear dependence of the apparent pseudo-first-order rate constants on DTT concentration. In the case of reduced polypeptide substrates, we assumed identical reactivities of all thiols in the beginning of the reaction (0-20% of final signal change), since DsbA randomly oxidizes polypeptides (14). To avoid errors due to decreased reactivities of buried thiols in folding intermediates, the initial velocities were used to calculate the apparent second-order rate constants for the reaction with hirudin and RNaseA.


RESULTS

Construction, Phenotypic Characterization, and Purification of DsbA Variants

We used the E. coli strain THZ2 lacking the chromosomal dsbA gene as host for expression and phenotypic characterization of the dsbA mutants. The in vivo activity of the dsbA mutants was tested in the absence of IPTG by the ability of the corresponding expression plasmids to complement the dsbA- phenotypes of E. coli THZ2. The strain THZ2 is immotile due to its inability to form disulfide bridges in the P-ring protein of the flagellar motor (30). Cells of E. coli THZ2 transformed with any of the plasmids containing dsbA mutants showed normal motility on soft agar comparable with the dsbA wild type gene. THZ2 also harbors a gene encoding the MalF-beta -galactosidase 102 fusion protein that only results in a Lac+ phenotype in the absence of disulfide bond formation in the periplasm (1, 3, 21). All dsbA mutants also restored disulfide bond formation in the periplasm and caused inactivation of beta -galactosidase. The in vivo activities of the dsbA mutants were not quantified further. The results of the assays were identical on rich medium and on minimal medium supplemented with all amino acids except cysteine and cystine. Hence, disulfide compounds of the growth medium can almost certainly be excluded as oxidants, and the DsbA variants are likely to be reoxidized by DsbB with an efficiency similar to the wild type.

DsbA WT and its variants were purified from periplasmic extracts by anion exchange chromatography on DE52-cellulose, hydrophobic chromatography on phenyl-Sepharose, and cation exchange chromatography on CM52-cellulose. The yields of the purified, oxidized protein varied from 13 to 19 mg of DsbA/liter of bacterial culture.

UV Absorbance, Fluorescence, and CD Spectra of DsbA Variants

UV absorbance spectra of native oxidized DsbA WT and all variants were identical and showed the same absorbance decrease at 280 nm upon denaturation in GdmCl.

The characteristic fluorescence properties of DsbA were also unchanged by any of the amino acid replacements. All variants still showed a more than 3-fold fluorescence increase at 330 nm (lambda ex = 295 nm) upon reduction of the active-site disulfide bridge and a shift in the emission maximum from 330 to 352 nm upon denaturation with GdmCl (12).

All DsbA variants were also indistinguishable from DsbA WT with respect to their far-UV and near-UV CD spectra. As observed for DsbA WT (19), the variants show the same characteristic change in their near-UV CD spectra upon reduction (data not shown).

Overall, the spectroscopic properties of all variants indicate that they are properly folded and that conformational changes due to the amino acid replacements do not influence the global structure.

Thermodynamic Stability of DsbA Variants

The influence of the amino acid replacements on protein stability (Delta Gstab) was determined by GdmCl-induced unfolding/refolding experiments (Fig. 2, Table I), which were followed by fluorescence measurements. For DsbA WT and all variants, cooperative and fully reversible transitions were observed, which were evaluated according to the two-state model of folding (32). Compared with DsbA WT, which is destabilized by its disulfide bond, replacement of Glu37 and/or Glu38 of the active-site helix by glutamine results in further destabilization of the oxidized form and stabilization of the reduced form. Hence, the energy difference between the oxidized and reduced form (Delta Delta Gstab) is increased in these variants.


Fig. 2. Reversible, GdmCl-dependent unfolding/refolding of oxidized and reduced DsbA WT and DsbA E37Q/E38Q at pH 7.0 and 25 °C. Oxidized WT (bullet , open circle ), reduced WT (black-square, square ), oxidized E37Q/E38Q (black-triangle, triangle ), and reduced E37Q/E38Q (black-down-triangle , down-triangle) are shown. Unfolding and refolding experiments are represented by closed symbols and open symbols, respectively. The fraction of unfolded molecules at equilibrium was calculated from the original data according to the two-state model (32).
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Table I.

Reversible unfolding/refolding of oxidized and reduced DsbA WT and DsbA variants in the presence of GdmCl

The transitions were measured at 25 °C in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA containing different concentrations of GdmCl and evaluated according to the two-state model of folding (32). Delta Delta Gstab is the stability difference between the native oxidized and the native reduced form (for details, see "Experimental Procedures").
Midpoint of transition Cooperativity  Delta Gstab  Delta Delta Gstab

M GdmCl kJ/mol·M GdmCl kJ/mol kJ/mol
WT 12.4  ± 5.5
  Oxidized 1.80 25.5  ± 1.6  -46.0  ± 2.9
  Reduced 2.37 24.6  ± 1.1  -58.4  ± 2.6
E37Q 20.6  ± 2.9
  Oxidized 1.79 21.8  ± 0.6  -39.1  ± 1.2
  Reduced 2.25 26.6  ± 0.7  -59.7  ± 1.7
E38Q 19.8  ± 3.2
  Oxidized 1.87 23.6  ± 0.6  -44.2  ± 1.2
  Reduced 2.44 26.2  ± 0.8  -64.0  ± 2.0
E37Q/E38Q 22.5  ± 2.8
  Oxidized 1.79 21.5  ± 0.5  -38.5  ± 1.0
  Reduced 2.30 26.6  ± 0.8  -61.0  ± 1.8
 Delta EVL 13.3  ± 4.6
  Oxidized 1.82 23.6  ± 1.4  -43.0  ± 2.6
  Reduced 2.24 25.2  ± 0.9  -56.3  ± 2.0
 Delta EVL/H41P 17.6  ± 4.6
  Oxidized 1.72 19.9  ± 0.9  -34.3  ± 1.6
  Reduced 2.19 23.7  ± 1.3  -51.9  ± 3.0

Interestingly, deletion of the tripeptide EVL does influence the stability of oxidized and reduced DsbA, but the additional exchange H41P also leads to an increase in Delta Delta Gstab (Table I).

Redox Properties of DsbA Variants

The thermodynamic cycle involving native oxidized, denatured oxidized, denatured reduced, and native reduced DsbA implies a correlation between the values of Delta Delta Gstab and the redox potentials of the variants (13, 19, 35). Hence, all variants should be more oxidizing than DsbA WT by 5-52 mV.

The redox properties of the DsbA variants were determined by measuring their redox equilibrium constants (Keq) with glutathione (12). Interestingly, none of the variants showed the expected decrease in Keq (Fig. 3, Table II). The variant E37Q is slightly more reducing than DsbA WT, whereas the redox potential of E38Q is increased by 11 mV. These effects are qualitatively additive in the double variant E37Q/E38Q. The redox potentials of Delta EVL and Delta EVL/H41P are decreased by 10 and 7 mV compared with DsbA WT, respectively.


Fig. 3. Redox equilibrium of DsbA WT (bullet ) and the variants E37Q (black-triangle), E38Q (black-down-triangle ), E37Q/E38Q (black-diamond ), Delta EVL (open circle ), and Delta EVL/H41P (square ) with glutathione at pH 7.0 and 25 °C. The fraction of reduced DsbA was determined by measuring the fluorescence at 332 nm in 100 mM sodium phosphate, 1 mM EDTA.
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Table II.

Redox properties and pKa values of the Cys30 thiol of DsbA WT and DsbA variants

Redox equilibrium constants (Keq) with glutathione were measured at 25 °C in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA containing 0.1 mM GSSG and 0.003-3 mM GSH. For calculation of the redox potential of DsbA, a value of -0.240 V (33) was used as the standard redox potential of GSH/GSSG. The pKa value of the thiol of Cys30 was determined at 25 °C as described in Fig. 4.
Keq E0' DsbA pKa of Cys30 SH

mM V
WT 0.126  ± 0.002  -0.125 3.40  ± 0.03
E37Q 0.144  ± 0.002  -0.126 3.69  ± 0.03
E38Q 0.055  ± 0.001  -0.114 3.52  ± 0.03
E37Q/E38Q 0.068  ± 0.001  -0.117 3.84  ± 0.02
 Delta EVL 0.276  ± 0.007  -0.135 3.92  ± 0.02
 Delta EVL/H41P 0.219  ± 0.006  -0.132 3.95  ± 0.04

Since negatively charged residues had been replaced by neutral residues in all these variants, the mutations may have led to a different electrostatic surrounding of the active-site disulfide. Therefore, the differences in their redox potential compared with DsbA WT should decrease upon increasing the ionic strength. In all cases, an increase in the sodium chloride concentration led to more reducing protein (Table III). The salt dependence of Keq is surprisingly low for DsbA WT and not changed in the variants. Therefore, we can exclude the possibility that negatively charged residues in the kink (Glu37, Glu38) have a large electrostatic effect on the active site of DsbA.

Table III.

Dependence on ionic strength of the redox equilibrium of DsbA WT and the DsbA variants with glutathione

Equilibrium constants (Keq) with glutathione were measured at 25 °C in 10 mM sodium phosphate, pH 7.0, 1 mM EDTA containing 0.1 mM GSSG, and 0.003-3 mM GSH and sodium chloride concentrations of 0, 0.2, 0.5, or 1.0 M. The relative increase of Keq with increasing ionic strength was calculated by dividing the respective values of Keq by the values of Keq at 0 M sodium chloride.
Sodium chloride concentration (M)
0 0.2 0.5 1.0

WT
  Keq (mM) 0.148  ± 0.002 0.213  ± 0.002 0.273  ± 0.004 0.369  ± 0.006
  Relative increase 1 1.44  ± 0.03 1.85  ± 0.05 2.50  ± 0.06
E37Q
  Keq (mM) 0.192  ± 0.002 0.331  ± 0.007 0.442  ± 0.008 0.608  ± 0.008
  Relative increase 1 1.72  ± 0.06 2.30  ± 0.07 3.17  ± 0.08
E38Q
  Keq (mM) 0.067  ± 0.001 0.102  ± 0.001 0.132  ± 0.001 0.184  ± 0.002
  Relative increase 1 1.52  ± 0.03 1.97  ± 0.04 2.73  ± 0.06
E37Q/E38Q
  Keq (mM) 0.082  ± 0.001 0.157  ± 0.002 0.236  ± 0.003 0.348  ± 0.006
  Relative increase 1 1.91  ± 0.04 2.87  ± 0.07 4.23  ± 0.12
 Delta EVL
  Keq (mM) 0.329  ± 0.008 0.510  ± 0.010 0.793  ± 0.021 1.027  ± 0.025
  Relative increase 1 1.55  ± 0.07 2.41  ± 0.12 3.12  ± 0.15
 Delta EVL/H41P
  Keq (mM) 0.306  ± 0.007 0.452  ± 0.009 0.689  ± 0.019 0.943  ± 0.023
  Relative increase 1 1.47  ± 0.07 2.25  ± 0.12 3.01  ± 0.15

pKa Values of the Cys30 Thiol

To test whether the amino acids in the kink of the active-site helix have any influence on the pKa value of the Cys30 thiol, the pH dependence of the fraction of the Cys30 thiolate was determined by its absorbance at 240 nm (18). All variants show slightly higher pKa values of the Cys30 thiol compared with DsbA WT, with the largest change of 0.5 pH units in the variants Delta EVL and Delta EVL/H41P (Fig. 4, Table II).


Fig. 4. Determination of the pKa of the thiol of Cys30 in DsbA WT (bullet ) and the variants E37Q (black-triangle), E38Q (black-down-triangle ), E37Q/E38Q (black-diamond ), Delta EVL (open circle ), and Delta EVL/H41P (square ) at 25 °C. The thiolate was detected by UV absorbance at 240 nm. Normalized transitions are shown. The solid lines correspond to fits according to the Henderson-Hasselbalch equation (see "Experimental Procedures" for details).
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In general, all mutations had a stronger influence on the stability of oxidized and reduced DsbA compared with changes in the pKa of Cys30. The interdependence of thermodynamic stability, redox potential, and the pKa value of Cys30 thiol therefore does not seem to be that simple, since we would have expected a lowered pKa value for the variants E38Q and E37Q/E38Q due to their increased redox potentials.

pH Dependence of Polypeptide Specificity

DsbA seems to be especially important for disulfide bond formation at acidic conditions, since it acts as oxidant even at pH 4 (14, 15). In addition, DsbA preferably reacts with reduced unfolded polypeptides compared with small organic thiols like the strong reductant dithiothreitol (14), which is presumably caused by a peptide binding site (14, 15, 16, 17). Using stopped-flow techniques we determined the pH dependence of DsbA's polypeptide specificity between pH 4 and 10. We used reduced unfolded RNase A and hirudin as polypeptide substrates. These proteins contain four and three disulfide bridges in their native forms, respectively. These polypeptide substrates were chosen because their isoelectric points (pIs) are strongly different (RNaseA pI = 9.6, (36); hirudin pI = 3.9, (37)). This allowed us to investigate whether DsbA's polypeptide specificity is influenced by the net charge of the substrate. In addition, both substrate proteins do not contain tryptophans. Therefore, oxidation of the substrates was followed by the increase in the tryptophan fluorescence of DsbA and evaluated according to apparent second-order reactions (see "Experimental Procedures" for details).

The rate constants of the reduction of DsbA WT and the variants by DTT are strongly dependent on pH and increase by 4 orders of magnitude from pH 4 to 10. In contrast, the rate constants of DsbA's reduction by both RNaseA and Hirudin increase by about 2 orders of magnitude between pH 4 and 6 but become nearly pH-independent between pH 6 and 10 (Fig. 5). Hence, the polypeptide specificity of DsbA and all variants is generally high under acidic conditions, decreases with increasing pH, and is independent of the pI of the polypeptide substrate. The negative charge of Glu37 appears to impair polypeptide binding slightly between pH 6 and 8, because the polypeptide specificities are significantly increased in the variants E37Q and E37Q/E38Q. However, none of the variants investigated showed a significantly altered pH dependence of polypeptide specificity. In addition, neither DsbA WT nor any of the variants exhibited a preference for the net charge of the substrate. Overall, we conclude that residues within or near the kink in the active-site helix of DsbA are not critical for the peptide specificity of the enzyme.


Fig. 5. pH dependence of the apparent second-order rate constants of the reduction of DsbA WT (bullet ) and E37Q/E38Q (black-diamond ) by DTT (solid line) and reduced, unfolded RNaseA (broken line) and hirudin (dotted line) at 25 °C. Measurements were followed fluorimetrically (lambda ex = 295 nm, lambda em >=  305 nm) on a stopped-flow instrument (see "Experimental Procedures" for details).
[View Larger Version of this Image (20K GIF file)]



DISCUSSION

Until now, biophysical studies on DsbA were restricted to the wild type protein (12, 13, 14, 18, 19) and variants where either the active-site cysteines (38, 39) or the XY dipeptide between them (21)3 had been exchanged.

The aim of this work was to investigate whether the unusual properties of DsbA's active-site disulfide are influenced by other regions of the protein, namely the kink in the active-site helix and residues of the acidic patch (7).

The in vivo activity of all variants reflects that they are catalytically active and can be reoxidized by the transmembrane protein DsbB, a reaction that involves formation of a DsbA/DsbB mixed disulfide (5, 6). The exact mode of interaction between DsbA and DsbB is still unclear, but if a specific recognition between the proteins existed that accelerates their disulfide exchange, Glu37 and residues 38-40 of DsbA would presumably not be involved in the critical contacts with DsbB.

A thermodynamic cycle has been used to explain the high redox potential of DsbA, which links the difference in stability of oxidized and reduced DsbA to the stability of the disulfide bond (redox potential) in the native protein (13, 19). As we observed an increase in Delta Delta Gstab for all variants investigated, we expected an increase in their intrinsic redox potentials. On the other hand, the observed increase of the pKa value of the Cys30 thiol in all variants should lead to a decrease in their redox potentials (21). Since the variants Delta EVL, Delta EVL/H41P, and E37Q have a lowered redox potential and the redox potential of the variants E38Q and E37Q/E38Q is increased, we could not confirm the predicted interdependence of redox potential, Delta Delta Gstab and pKa of the Cys30 thiol. Obviously, the mechanism underlying the high redox potential of DsbA is more complex.3 One additional parameter may be the pKa of the Cys33 thiol, which has been assumed to be constant in previous model studies (21) but which might also change as a result of the mutations.

Electrostatic calculations had suggested that removal of Glu37 or Glu38 might further increase the redox potential of DsbA by 50 mV (23). However, only a slight increase of 11 mV was observed for the E38Q variant, and no change in redox properties could be detected for E37Q. E38Q and E37Q/E38Q are the first variants of DsbA that are more oxidizing than the wild type protein. Obviously, the charged side chains of Glu37 and Glu38 are not critical determinants of the oxidative force of DsbA.

DsbA accelerates thiol-disulfide exchange reactions even at acidic pH (14, 15) and oxidizes polypeptide substrates faster than small dithiol compounds such as DTT (14). This property can be explained by an additional noncovalent interaction between DsbA and the polypeptide substrate and suggests the existence of a polypeptide binding site (7, 16, 17). We show here that this peptide specificity is independent of the isoelectric point of the polypeptide substrate but strongly dependent on pH, with a higher specificity at low pH. Since only the thiolate acts as a nucleophile, the rate constants of thiol-disulfide exchange reactions strongly increase with increasing pH below the pKa of the attacking thiol (20). A higher substrate specificity with decreasing pH thus means that the rate of oxidation of polypeptide substrates is less pH-dependent than the rate of oxidation of small thiol compounds. This fact might become important in vivo for disulfide bond formation at acidic growth conditions. The growth limit for E. coli under acidic conditions is pH 4.4 (40), where spontaneous disulfide exchange reactions no longer occur.

Since the polypeptide specificity is not strongly influenced in any of the variants tested, we can almost certainly exclude that the kink is part of the proposed peptide binding site in DsbA.

It is generally accepted that the interactions of alpha -helix dipoles with charged residues are of functional and structural importance (41), and they have been shown to influence protein stability (42, 43, 44, 45). The partial positive charge at the amino terminus of the active-site helices of disulfide oxidoreductases has recently been claimed to stabilize the negative charge of the nucleophilic cysteine thiolate and thereby lower its pKa (22). Removal of the tripeptide Glu38-Val39-Leu40 and the additional replacement H41P was not expected to cause exactly the conformational change of the active-site helix of DsbA to the conformations found in glutaredoxin and thioredoxin, respectively. However, we at least expected changes in the physicochemical properties of DsbA in the direction of glutaredoxins and thioredoxins. Indeed, we observed slightly increased pKa values of Cys30 thiols and slightly lowered redox potentials (Table II) for both variants. These differences are small, however, compared with the effects reported for mutations in the XY dipeptide in DsbA (21). It thus appears that residues in the amino-terminal part of the active-site helices of disulfide oxidoreductases have a much stronger influence on their redox properties than residues in the carboxyl-terminal part. The almost unchanged properties of the Delta EVL variant and the Delta EVL/H41P double variant are in accordance with the fact that the tripeptide insertion is absent and that a proline equivalent to residue 41 in DsbA is present in the DsbA-related protein from Vibrio cholerae (46).

Since the alpha -helix dipole causes a partial negative charge at the carboxyl terminus of the helix, the charged residues Glu37 and Glu38 at the carboxyl terminus of the first part of the active-site helix should be energetically unfavorable. In addition, both residues are part of the acidic patch and are thus located in a strongly, negatively charged environment (7). The increased stability of the reduced variants E37Q, E38Q, and E37Q/E38Q compared with DsbA WT is in accordance with this consideration. However, the origin of the decreased stability of their oxidized forms remains unclear. Overall, the stability changes are caused by changes in cooperativity rather than by changes in the midpoint of transition. In principle, replacement of energetically unfavorable, charged residues that cause repulsive interactions within the protein might lead to a more compact structure and thus to a higher cooperativity of the unfolding transitions compared with DsbA WT. However, this was only observed for the reduced forms of the variants. In contrast, the cooperativities were unexpectedly decreased for the oxidized forms. This could mean that the assumed two-state model of folding is no longer strictly valid for the oxidized variants (47). Until now, only the three-dimensional structure of DsbA's oxidized form is known (7). Therefore, no information on structural changes that occur upon reduction of DsbA is available. We speculate that the opposite effects of the amino acid replacements on the unfolding/refolding transitions of DsbA might indeed reflect conformational differences in the oxidized and reduced form. However, three-dimensional structures of the oxidized and reduced forms of E. coli glutaredoxin (48), E. coli thioredoxin (49), and human thioredoxin (50, 51) have revealed that only minor structural differences exist between the redox states of these enzymes.

In conclusion, we have shown that neither Glu37, Glu38, nor the kink itself has a large influence on the physicochemical properties of DsbA, such as the pKa value of the Cys30 thiol or the redox potential. It appears that the properties of DsbA are largely determined by the PH dipeptide in the active-site CPHC, which may strongly influence the helix-dipole. We could demonstrate that Glu37 and Glu38, which are part of an unusual acidic patch on the surface of DsbA, do not contribute to its peptide specificity and are not critical for reoxidation of DsbA by DsbB.


FOOTNOTES

*   This work was supported by a research grant from the Eidgenössische Technische Hochschule Hönggerberg, Zürich. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: +41-1-633-6819; Fax: +41-1-633-1036; E-mail: rudi{at}mol.biol.ethz.ch.
1    The abbreviations used are: DTT, 1,4-dithio-DL-threitol; IPTG, isopropyl-beta -D-thiolgalactoside; MOPS, 4-morpholinepropanesulfonic acid; WT, wild type; GdmCl, guanidinium chloride.
2    A. Jacobi and R. Glockshuber, manuscript in preparation.
3    M. Huber-Wunderlich and R. Glockshuber, manuscript in preparation.

Acknowledgments

We thank M. Huber-Wunderlich and A. Jacobi for fruitful discussions, V. Eggli for help during construction of the DsbA mutants, G. Frank for Edman sequencing, and P. James for performing electrospray mass spectroscopy.


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