Elimination of All Charged Residues in the Vicinity of the Active-site Helix of the Disulfide Oxidoreductase DsbA
INFLUENCE OF ELECTROSTATIC INTERACTIONS ON STABILITY AND REDOX PROPERTIES*

(Received for publication, April 7, 1997, and in revised form, June 19, 1997)

Alexander Jacobi Dagger , Martina Huber-Wunderlich , Jens Hennecke and Rudi Glockshuber

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 alpha -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 (Delta Delta 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 Delta Delta Gox/red values, demonstrating that the relationship between these parameters is far more complex than previously thought.


INTRODUCTION

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 alpha -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 alpha -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 alpha -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).


Fig. 1. Ribbon diagram of the three-dimensional structure of oxidized DsbA from E. coli (17). The thioredoxin-like domain and the helical domain of DsbA are shown in light and dark gray, respectively. The active-site cysteine residues and the side chains of all residues exchanged in this study are indicated by ball-stick representation. The sulfur atoms of the active-site disulfide are depicted in black. The figure was created with the program MOLMOL (66).
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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.


Fig. 2. Structure-based alignment of the known DsbA sequences and sequences of thioredoxins and PDIs in the region of the active-site helix. The active-site cysteines and the residues of the conserved salt bridge corresponding to Glu24 and Lys58 in DsbA are shown in boldface type. Residues that were exchanged in this study are marked by asterisks. The positions of active-site helices and the adjacent beta -strands are inferred from the structures of E. coli thioredoxin (30), E. coli DsbA (17), and the a domain of human PDI (9). Shown are sequences from E. coli, Shigella flexneri, Erwinia chrysanthemi, Hemophilus influenzae, Vibrio chloerae, Azotobacter vinelandii, Legionella pneumophilia, Salmonella enteritidis, Salmonella typhimurium, and Salmonella typhi.
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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 epsilon -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.


EXPERIMENTAL PROCEDURES

Materials

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-beta -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-beta -D-galactopyranoside (X-gal), and polymyxin B sulfate were purchased from Sigma-Aldrich (Deisenhofen, Germany). All other chemicals were of analytical grade.

Plasmid Construction and Mutagenesis

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).

Overproduction and Purification of DsbA Variants

Cells of E. coli THZ2 (dsbA::kan, recA::cam, lambda 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).

Detection of DsbA Activity in Vivo

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. beta -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 Techniques

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 Equilibria

Unfolding/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 Potentials

For 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.
R=([<UP>GSH</UP>]<SUP>2</SUP>/[<UP>GSSG</UP>])/(K<SUB><UP>eq</UP></SUB>+[<UP>GSH</UP>]<SUP>2</SUP>/[<UP>GSSG</UP>]) (Eq. 1)

Determination of the pKa Value of Cysteine 30

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 beta -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,
S=S<SUB><UP>AH</UP></SUB>+(S<SUB><UP>A</UP><SUP><UP>-</UP></SUP></SUB>−S<SUB><UP>AH</UP></SUB>)/(1+10<SUP><UP>p</UP>K<SUB>a</SUB><UP>−pH</UP></SUP>)<UP>,</UP> (Eq. 2)
in which SAH is the corrected signal of the fully protonated and SA- is that of the fully deprotonated form. Evaluation of the pKa of Cys30 of DsbA yielded reliable results between pH 7.8 and 2.2, and further acidification resulted in denaturation of DsbA. Deviations above pH 7.8 may have resulted from increasing deprotonation of tyrosine residues.

Investigation of Polypeptide Specificity

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.


RESULTS

Construction and Purification of DsbA Variants

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 THZ2

We 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-beta -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 beta -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.

Spectroscopic Properties of the DsbA Variants

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 Variants

The 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).


Fig. 3. GdmCl-dependent folding/unfolding equilibria of oxidized and reduced wild type DsbA and the variant E24Q at pH 7.0 and 25 °C. Open symbols (open circle , square , diamond , down-triangle) correspond to unfolding experiments, and closed symbols (bullet , black-square, black-diamond , black-down-triangle ) correspond to refolding experiments. open circle  and bullet , oxidized DsbA wild type; diamond  and black-diamond , reduced DsbA wild type; square  and black-square, oxidized variant E24Q; down-triangle and black-down-triangle , reduced variant E24Q. Only one-half of each set of data points is shown. The normalized transitions were obtained from the original fluorescence data after a six-parameter fit (solid lines), assuming the two-state model of folding (47). The thermodynamic stabilities of all oxidized and reduced variants studied are summarized in Table I.
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Table I. Thermodynamic stabilities (Delta GStab) of oxidized and reduced wild type DsbA and the DsbA variants at 25 °C and pH 7.0

GdmCl-dependent unfolding/refolding equilibria were measured as described in the legend to Fig. 3. Delta Delta GWT corresponds to the difference between Delta GStab of an oxidized or reduced DsbA variant and the corresponding redox form of the wild type (Delta GStab(variant) - Delta GStab(wild type)). Delta Delta Gox/red represents the difference between the free energy of folding of the oxidized and the reduced forms of a DsbA variant. An entropic destabilization of the oxidized, unfolded proteins by the disulfide bond was not considered.

DsbA Transition midpoint Cooperativity  Delta GStab  Delta Delta GWT  Delta Delta Gox/red

M GdmCl kJ/mol · M kJ/mol kJ/mol kJ/mol
Wild type 10.2  ± 5.0
  Oxidized 1.82 26.3  ± 1.8  -47.9  ± 2.7
  Reduced 2.39 24.3  ± 1.0  -58.1  ± 2.3
E24Q 10.1  ± 3.7
  Oxidized 1.88 26.7  ± 0.9  -50.2  ± 1.7  -2.3  ± 4.4
  Reduced 2.43 24.8  ± 0.8  -60.3  ± 2.0  -2.2  ± 4.3
K58M 21.5  ± 4.9
  Oxidized 1.83 21.6  ± 0.8  -39.6  ± 1.5 +8.3  ± 4.2
  Reduced 2.38 25.7  ± 1.4  -61.1  ± 3.4  -3.0  ± 5.7
E37Qa 20.6  ± 2.9
  Oxidized 1.79 21.8  ± 0.6  -39.1  ± 1.2 +8.8  ± 3.9
  Reduced 2.25 26.6  ± 0.7  -59.7  ± 1.7  -1.6  ± 4.0
E24Q/K58M 8.3  ± 6.7
  Oxidized 1.90 27.2  ± 2.3  -51.8  ± 4.3  -3.9  ± 7.0
  Reduced 2.38 25.2  ± 1.0  -60.1  ± 2.4  -2.0  ± 4.7
E24Q/E37Q 7.6  ± 5.3
  Oxidized 1.82 23.3  ± 1.4  -42.5  ± 2.5 +5.4  ± 5.2
  Reduced 2.26 22.2  ± 1.2  -50.1  ± 2.8 +8.0  ± 5.1
E37Q/K58M 24.9  ± 3.3
  Oxidized 1.76 19.7  ± 0.7  -34.6  ± 1.3 +13.3  ± 4.0
  Reduced 2.23 26.7  ± 0.9  -59.5  ± 2.0  -1.4  ± 4.3
E24Q/E37Q/K58M 15.2  ± 4.6
  Oxidized 1.85 22.5  ± 1.0  -41.7  ± 1.8 +6.2  ± 4.5
  Reduced 2.36 24.1  ± 1.2  -56.9  ± 2.8 +1.2  ± 5.1
E24Q/E37Q/E38Q/K58M  -7.2  ± 4.4b
  Oxidized 1.88 22.3  ± 1.3  -42.0  ± 2.4 +5.9  ± 5.1
  Reduced 2.29 15.2  ± 0.9b  -34.8  ± 2.0b +23.3  ± 4.3b

a Adapted from Hennecke et al. (20).
b Two-state model of folding may not be valid.

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.

Table II. Double mutant cycle analysis of contributions of the interactions Glu24-Lys58, Glu37-Lys58, and Glu24-Glu37 to the thermodynamic stability of oxidized and reduced DsbA

Delta Delta GWT, Delta Delta GE37Q, Delta Delta GE24Q, and Delta Delta GK58M correspond to the difference between Delta GStab of an oxidized or reduced DsbA variant and the corresponding redox form of the wild type and the variants E37Q, E24Q, and K58M, respectively. The corresponding delta  values represent the energy by which a side chain-side chain interaction stabilizes or destabilizes the protein and is calculated from delta  = Delta Delta Gdouble variant - (Delta Delta Gvariant 1 + Delta Delta Gvariant 2).

DsbA Oxidized
Reduced
Oxidized
Reduced
 Delta Delta GWT  delta WT  Delta Delta GWT  delta WT  Delta Delta GE37Q  delta E37Q  Delta Delta GE37Q  delta E37Q

kJ/mol kJ/mol kJ/mol kJ/mol
E24Q  -2.3  -2.2  -11.1  -0.6
K58M  +8.3  -3.0  -0.5  -1.4
E24Q/K58M  -3.9  -9.9  -2.0 +3.2  -12.7  -2.1  -0.4 +1.6
 Delta Delta GE24Q  delta E24Q  Delta Delta GE24Q  delta E24Q

E37Q  +8.8  -1.6 +11.1 +0.6
K58M  +8.3  -3.0 +10.6  -0.8
E37Q/K58M +13.3  -3.8  -1.4 +3.2 +15.6  -6.1 +0.8 +1.0
 Delta Delta GK58M  delta K58M  Delta Delta GK58M  delta K58M

E24Q  -2.3  -2.2  -10.6 +0.8
E37Q  +8.8  -1.6 +0.5 +1.4
E24Q/E37Q  +5.4  -1.1 +8.0 +11.8  -2.9 +7.2 +11.0 +8.8
E24Q  -2.3  -2.2
E37Q  +8.8  -1.6
K58M  +8.3  -3.0
E24Q/E37Q/K58M  +6.2  -8.6 +1.2 +8.0

Overall, the measured stability differences between the oxidized and reduced variants (Delta Delta 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).

Comparison of Measured and Predicted Redox Potentials of the DsbA Variants

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 (Delta Delta 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 Delta Delta 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.


Fig. 4. Redox equilibria of DsbA wild type (bullet ) and the variants E24Q (open circle ), E24Q/E37Q (black-down-triangle ), and E37Q/K58M (down-triangle) with glutathione at pH 7.0 and 25 °C. The fraction of reduced protein at equilibrium (R) was measured by the redox state-dependent tryptophan fluorescence of DsbA at 330 nm (excitation at 280 nm). The solid lines correspond to the fits of the normalized fluorescence data according to Equation 1 (see "Experimental Procedures"). The equilibrium constants with glutathione and the redox potentials for all investigated DsbA variants are given in Table III.
[View Larger Version of this Image (23K GIF file)]

Table III. Measured pKa values of the Cys30 thiol, equilibrium constants with glutathione, and redox potentials of DsbA wild type and the DsbA variants at 25 °C and comparison with predicted redox potentials

The pKa values of the thiol of Cys30 were determined by absorbance measurements at 240 nm as described in Fig. 5. Equilibrium constants (Keq) with glutathione were measured at pH 7.0 as described in the legend to Fig. 4. A value of -240 mV (53) was used as the standard redox potential of GSH/GSSG to calculate the redox potential of DsbA. The changes in redox potential compared with the wild type (Delta E'0 WT) were predicted from the measured pKa values of the Cys30 thiol according to the theory of Szajewski and Whitesides (54) assuming a constant pKa of Cys33 of 9.5. Delta E'0 WT was also predicted from Delta Delta Delta Gox/red, the energy difference between Delta Delta Gox/red of a DsbA variant and Delta Delta Gox/red of DsbA wild type (see Table I). In both cases Delta E'0 is obtained from the equation Delta E'0 = -(RT/2F) · ln(Kvariant/KWT), with Kvariant and KWT representing the predicted equilibrium constants with glutathione of a DsbA variant and the wild type, respectively.

DsbA pKa of the Cys30 thiol Keq E'0  Delta E'0 WT (measured)  Delta E'0 WT predicted from Delta Delta Delta Gox/red  Delta E'0 WT predicted from pKa of Cys30 thiol

mM mV mV mV mV
Wild type 3.55  ± 0.02 0.108  ± 0.002  -123
E24Q 3.52  ± 0.03 0.087  ± 0.002  -120 +3  -1 +1
K58M 3.19  ± 0.06 0.082  ± 0.002  -119 +4 +59 +13
E37Qa 3.69  ± 0.03 0.144  ± 0.002  -126  -3 +54  -5
E24Q/K58M 4.46  ± 0.06 0.184  ± 0.004  -130  -7  -10  -32
E24Q/E37Q 3.81  ± 0.03 0.052  ± 0.001  -113 +10  -14  -9
E37Q/K58M 3.50  ± 0.05 0.210  ± 0.002  -131  -8 +77 +2
E24Q/E37Q/K58M 3.94  ± 0.06 0.110  ± 0.002  -123 0 +26  -14
E24Q/E37Q/E38Q/K58M 3.89  ± 0.05 0.066  ± 0.002  -116 +7  -12

a Adapted from Hennecke et al. (20).

Ionization of the Cysteine 30 Thiol

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.


Fig. 5. Ionization of the Cys30 side chain of reduced DsbA wild type (bullet ) and the reduced variants E24Q (open circle ), K58M (black-down-triangle ), and E24Q/K58M (black-down-triangle ) at 25 °C. The ionization of the Cys30 side chain was detected by the specific absorbance of the thiolate anion at 240 nm. The corresponding oxidized proteins were used as a reference. The corrected data were fitted according to the Henderson-Hasselbalch equation (Equation 2) (solid lines) and normalized. The pKa values of the Cys30 side chains of all variants investigated are listed in Table III.
[View Larger Version of this Image (21K GIF file)]

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).


Fig. 6. pH dependence of the polypeptide specificity of wild type DsbA and the DsbA variants at 25 °C. The polypeptide specificity of DsbA was defined as the ratio between the apparent second-order rate constant of the random oxidation of a reduced substrate protein and the apparent second-order rate constant of oxidation of DTT (kprotein/kDTT). Reduced RNase A (A) and reduced hirudin (B) were used as examples for proteins with basic and acidic pI, respectively. The reactions were performed under pseudo-first-order conditions and followed by the increase in tryptophan fluorescence caused by the generation of reduced DsbA (for details see "Experimental Procedures"). bullet , DsbA wild type; diamond , E24Q; open circle , K58M; square , E24Q/K58M; black-diamond , E37Q/K58M; black-square, E24Q/E37Q; black-triangle, E24Q/E37Q/K58M; black-down-triangle , E24Q/E37Q/E38Q/K58M.
[View Larger Version of this Image (25K GIF file)]


DISCUSSION

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 alpha -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).

Thermodynamic Stabilities of the DsbA Variants and Function of Glu24

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),
&Dgr;&Dgr;G(<UP>pH</UP>)=<UP>−</UP>RT <UP>ln</UP>[(1+10<SUP><UP>pH</UP>−<UP>p</UP>K<SUB>a(<UP>f</UP>)</SUB></SUP>)/(1+10<SUP><UP>pH</UP>−<UP>p</UP>K<SUB>a(<UP>u</UP>)</SUB></SUP>)] (Eq. 3)
in which Delta Delta G is the energy by which the acidic residue stabilizes or destabilizes the protein, and pKa (f) and pKa (u) are the pKa values of the acidic residue in the native and unfolded protein, respectively.

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 Delta Delta 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 Delta Delta 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 Potentials

In principle, the following predictions can be made for the relationship between the difference of the thermodynamic stabilities of the oxidized and reduced form (Delta Delta Gox/red), the pKa of the Cys30 thiol, and the equilibrium constant with glutathione (redox potential) of DsbA. (i) The Delta Delta 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 (Delta E0' WT) (18, 19). (iii) Delta Delta Gox/red should also directly correlate with Delta 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 Delta Delta 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 Delta Delta 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 Delta Delta 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 Delta Delta Gox/red, E0', and the pKa of the Cys30 thiol.

In Vivo Activity and Polypeptide Specificity of the DsbA Variants

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 Bridge

The 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.


FOOTNOTES

*   This work was supported by Grant  JA 761/1-1 from the Deutsche Forschungsgemeinschaft (to A. J.) and a grant from the Eidgenössische Technische Hochschule-Hönggerberg Zürich (to R. G.).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.: 41-1-633-3274; Fax: 41-1-633-1036; E-mail: jacobi{at}mol.biol.ethz.ch.
1   The abbreviations used are: PDI, protein disulfide isomerase; Bicine, N,N-bis(2-hydroxyethyl)glycine; DTT, 1,4-dithio-DL-threitol; GdmCl, guanidinium chloride; IPTG, isopropyl-beta -D-thiogalactopyranoside; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; X-gal, 5-bromo-4-chloro-3-indolyl-beta -Dgalactopyranoside.
2   T. Zander, unpublished data.

ACKNOWLEDGEMENTS

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.


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