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Originally published In Press as doi:10.1074/jbc.M311833200 on November 3, 2003

J. Biol. Chem., Vol. 279, Issue 5, 3516-3524, January 30, 2004
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Gram-positive DsbE Proteins Function Differently from Gram-negative DsbE Homologs

A STRUCTURE TO FUNCTION ANALYSIS OF DsbE FROM MYCOBACTERIUM TUBERCULOSIS*

Celia W. Goulding{ddagger}, Marcin I. Apostol{ddagger}, Stefan Gleiter§, Angineh Parseghian||, James Bardwell§**, Marila Gennaro{ddagger}{ddagger}, and David Eisenberg{ddagger}||§§

From the {ddagger}Howard Hughes Medical Institute and UCLA-Department of Energy Institute of Genomics and Proteomics, Los Angeles, California 90095-1570, the ||Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1570, the §Department of Molecular Cellular and Development Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, and the {ddagger}{ddagger}Public Health Research Institute, Newark, New Jersey 07103

Received for publication, October 29, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacterium tuberculosis, a Gram-positive bacterium, encodes a secreted Dsb-like protein annotated as Mtb DsbE (Rv2878c, also known as MPT53). Because Dsb proteins in Escherichia coli and other bacteria seem to catalyze proper folding during protein secretion and because folding of secreted proteins is thought to be coupled to disulfide oxidoreduction, the function of Mtb DsbE may be to ensure that secreted proteins are in their correctly folded states. We have determined the crystal structure of Mtb DsbE to 1.1 Å resolution, which reveals a thioredoxin-like domain with a typical CXXC active site. These cysteines are in their reduced state. Biochemical characterization of Mtb DsbE reveals that this disulfide oxidoreductase is an oxidant, unlike Gram-negative bacteria DsbE proteins, which have been shown to be weak reductants. In addition, the pKa value of the active site, solvent-exposed cysteine is ~2 pH units lower than that of Gram-negative DsbE homologs. Finally, the reduced form of Mtb DsbE is more stable than the oxidized form, and Mtb DsbE is able to oxidatively fold hirudin. Structural and biochemical analysis implies that Mtb DsbE functions differently from Gram-negative DsbE homologs, and we discuss its possible functional role in the bacterium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein disulfide bonds are often buried and serve to stabilize protein structures. However, partially exposed disulfide bonds in the active sites of protein-disulfide oxidoreductases have a variety of mechanistic roles in protein folding, electron transport, and bioenergetics in organisms ranging from prokaryotes to humans. Most disulfide oxidoreductases contain a conserved thioredoxin-like domain such as in thioredoxin, eukaryotic protein disulfide bond isomerase, glutaredoxin (1), peroxiredoxins (2), and disulfide bond-forming proteins (Dsb).1 Except for peroxiredoxins, all of these proteins share a common sequence motif (CXXC) at their active sites.

One such family of disulfide oxidoreductases is the Dsb proteins, which are best characterized in Escherichia coli. These proteins reside in the periplasmic space of Gram-negative bacteria (Fig. 1a) and are necessary for the correct folding of many cell envelope proteins (3). E. coli DsbE is a thioredoxin-like protein, involved in cytochrome c maturation (4). DsbE has been implicated in the reduction of the thiol ether linkers to the heme of apocytochrome c (5), prior to heme ligation by CcmF and CcmH (4, 6). E. coli DsbD is a cytoplasmic transmembrane protein responsible for maintaining DsbE in its reduced state in the periplasm (7). E. coli DsbC is a homodimer with disulfide bond isomerase activity that is also kept reduced by the transmembrane protein DsbD (8, 9). In contrast, E. coli DsbA is a monomer that catalyzes the oxidation of reduced, unfolded proteins (10, 11). DsbA is reoxidized by the transmembrane protein DsbB, which is in turn oxidized by components of the electron transport pathway (12, 13). Dsb proteins, in particular DsbA, have been shown to be involved in virulence in toxin-secreting Gram-negative bacteria such as Vibrio cholerae (14, 15), Yersinia pestis (16), Shigella sp. (17), and E. coli (18). Gram-positive bacteria do not have a periplasm, and proteins that are secreted from the cytoplasm are either cell wall-associated or extracellular. In Mycobacterium tuberculosis, the only Dsb proteins present are homologs to E. coli DsbE (Mtb DsbE or Rv2878c, also known as MPT53) and its redox, transmembrane protein partner, E. coli DsbD (Mtb DsbD or Rv2874), which are depicted in Fig. 1b. The presence of Dsb proteins in Gram-positive bacteria, such as M. tuberculosis, suggests that these proteins are necessary for the correct folding of cell wall-associated and extracellular secreted proteins. Hence, studies of Mtb DsbE may give some insights into the virulence of mycobacteria.



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  		FIG. 1.
Model for disulfide bond formation and rearrangement of catalyzed by Dsb proteins of E. coli (a) and M. tuberculosis (b). a, known Dsb proteins in the E. coli (Gram-negative bacteria) system. DsbA oxidizes disulfide bonds of newly translocated proteins. The transmembrane protein DsbB accepts electrons from DsbA and transfers them to quinone (Q) or menaquinones (MQ) embedded in the bilayer. DsbC is a disulfide isomerase protein that catalyzes the reformation of incorrectly formed disulfide bonds. DsbE is thought to reduce the cysteines of apocytochrome c for heme attachment and is anchored to the inner membrane. These two proteins accept electrons from the cytoplasm (generated from NADPH, thioredoxin (TrxA), and thioredoxin reductase (TrxB)) through the DsbD transmembrane protein. The Dsb proteins all reside in the periplasm of Gram-negative bacteria. b, model of proposed Dsb proteins in the M. tuberculosis (Gram-positive bacteria) system. The Dsb protein homologs to the E. coli Dsb system found in M. tuberculosis reside in the extracellular space (i.e. these proteins are secreted from the bacterial cell wall into the extracellular space). Mtb DsbE, which is homologous to E. coli DsbE protein, has a cleavable signal peptide. Mtb DsbD (Rv2874) is homologous to the E. coli DsbD transmembrane protein, which has two predicted soluble domains on the N and C termini of the protein that are connected by eight "predicted" transmembrane helices.

 
M. tuberculosis is a pathogenic bacterium that is responsible for tuberculosis, which is the world's leading cause of adult death by a bacterial infectious disease, with 3 million deaths and 10 million new cases per year (19, 20). The M. tuberculosis Structural Genomics Consortium is an international effort focusing on determining protein structures from M. tuberculosis, including potential drug targets (21, 22). A promising group of potential drug targets is the secreted proteins of M. tuberculosis, which are important in the induction and escape from host immune responses and virulence. Mtb DsbE is one such uncharacterized secreted protein. This work presents an example of protein structural analysis leading to functional information.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Purification and Crystallization—A recombinant plasmid producing the mature DsbE (without the signal peptide, residues 30-159) in pQE30 (Qiagen) was constructed as described previously (23). The purification of Mtb DsbE has been previously described (24). The purified protein was dialyzed into 0.5 M NaCl and 0.1 M Tris·HCl, pH 7.4, for crystallization trials. The protein crystallized in 2.2 M NH4SO4, 5% isopropyl alcohol, 20% glycerol; the crystals were mounted; and diffraction data were collected under cryoconditions identical to the crystallization conditions. The selenomethionine Mtb DsbE protein was prepared as previously described (25) and crystallized under identical conditions to the native protein.

Data Collection and Structure Determination and Refinement—A selenomethionine-substituted Mtb DsbE crystal diffracted to 1.5 Å, and a native crystal diffracted to 1.1 Å, both having unit cell dimensions of 60.7 x 60.7 x 80.0 Å with one monomer per asymmetric unit in space group P43212. Data were processed using DENZO and SCALEPACK (26) and multiwavelength anamolous diffraction phasing proceeded by the usual methods of heavy atom location (SHELDX, available on the World Wide Web at shelx.uni-ac.gwdg.de/SHELX/, maximum likelihood phase refinement (27), and density modification (28)). Phase extension to 1.1 Å permitted automated model building for all but five residues of the protein with ARP/WARP (29) and was refined using SHELXL. Model building was done in O (30), and the final model includes all 134 residues of the monomer. Data and refinement statistics are shown in Table I.


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  		TABLE I
X-ray diffraction data collection and atomic refinement for disulfide oxidoreductase DsbE protein from M. tuberculosis The final column refers to the native protein and the receding columns to the selenomethionine analog.

 
Structure and Sequence Analysis—BLAST and ClustalW were used for data base searches and multiple sequence alignments, respectively. Pairwise alignments were calculated by the Smith-Waterman algorithm. Similar protein structures were searched for using DALI (31) and aligned with combinatorial extension (32). Electrostatic and surface area calculations were performed with WebLabViewer.

Oxidation and Reduction of Mtb DsbE—To oxidize Mtb DsbE, 10 mM GSSG was added to Mtb DsbE in 0.5 M NaCl and 0.1 M Tris·HCl, pH 7.4, and incubated for 1 h at room temperature. The oxidized protein was then isolated by gel filtration in its original buffer. To reduce Mtb DsbE, 100 mM dithiothreitol was added to Mtb DsbE in 0.5 M NaCl and 0.1 M Tris·HCl, pH 7.4, and incubated overnight at 4 °C. The reduced protein was then isolated by gel filtration in its original buffer. To ensure total reduction or oxidation, samples of the treated Mtb DsbE were run on an SDS-PAGE gel with no dithiothreitol present, because oxidized samples migrate faster than reduced samples under nonreducing conditions.

Redox Properties of Mtb DsbE: Comparison with Glutathione—The in vitro redox state of Mtb DsbE was assayed as described (33, 34). In this assay, the change in fluorescence intensity (excitation wavelength 280 nm) was measured at the wavelength of maximum emission (356 nm for Mtb DsbE). Experiments were carried out in 100 mM sodium phosphate, pH 7.0, and 1.0 mM EDTA. Oxidized and reduced Mtb DsbE were incubated at 25 °C in the presence of 0.1 mM GSSG and varying concentrations of GSH (0-10 mM) for 12 h before recording the fluorescence emission on a Spex Fluorolog (Jubin Yvon-Spex). The equilibrium concentrations of GSH and GSSG were calculated according to Equations 1, 2, 3,

(Eq. 1)

(Eq. 2)

(Eq. 3)
where [GSH]0 and [GSSG]0 represent the initial concentrations of GSH and GSSG, R is the relative amount of reduced protein at equilibrium, [DsbE]0 is the initial concentration of Mtb DsbE in the oxidized form, F is the fluorescence intensity, and Fox and Fred are the fluorescence intensities of completely oxidized and reduced protein. The equilibrium constant Keq was estimated from nonlinear regression analysis of the data according to the Nernst equation (Equation 4).

(Eq. 4)
From the equilibrium constant and by using the glutathione standard potential (E'0 GSH/GSSG = -240 mV) (35), the standard redox potential (E'0) was calculated with Equation 5.

(Eq. 5)
in which F represents Faraday's constant, n is the number of electrons transferred, and RT is the product of the gas constant and the absolute temperature.

Redox Properties of Mtb DsbE: Comparison with Disulfide Bond Isomerase Protein, DsbC—The redox potential of Mtb DsbE was determined by the method of protein-protein redox equilibria developed by Aslund (35), as modified by Collet and Bardwell (36). The protein used as a standard was E. coli DsbC, which has a redox potential (E'0)of -135 mV (37). In short, equimolar amounts of oxidized DsbC and reduced Mtb DsbE were incubated in 50 mM Tris/HCl, pH 7.5, 350 mM NaCl, 5 mM EDTA at 25 °C. After different time points, samples were analyzed by reverse phase high pressure liquid chromatography (2695 separations module; Waters). The oxidized and reduced forms of each protein were separated on a Phenomenex Primesphere 5-µm C8 column (buffer A: 0.1% trifluoroacetic acid in water; buffer B: 30% methanol, 60% acetonitrile, 0.1% trifluoroacetic acid). Separation of oxidized and reduced forms of DsbC and Mtb DsbE was achieved using a linear gradient from 53 to 62% B over 450 min at a flow rate of 1 ml/min. For evaluation of the redox potential, peak areas were analyzed using PeakFit (Systat), and the redox potential was calculated as described previously (35).

Determination of pKa of Cys36—The pH-dependent ionization of the Cys36 thiol (solvent-exposed) was followed by the specific absorbance of the thiolate anion at 240 nm as described earlier (9). As a control, the pH-dependent absorbance for the oxidized form of Mtb DsbE was recorded. To avoid precipitation artifacts and to minimize buffer absorbance, a buffer system consisting of 10 mM K2PO4, 10 mM boric acid, 10 mM sodium succinate, 1 mM EDTA, and 200 mM KCl (containing 100 µM dithiothreitol for the reduced protein) was used. The pH (initial value of 8.5) was lowered to 2.2 by the 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 ~30 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 (Equation 6), in which SAH represents the corrected absorption intensity of the fully protonated form, and SA - is that of the fully deprotonated form.

(Eq. 6)

Determination of Unfolding/Folding Equilibrium—The reversible guanidine hydrochloride (GdnHCl)-induced unfolding/folding of Mtb DsbE was performed by measuring the CD ellipticities at 222 nm (38). The spectrum of the reduced form was recorded in the presence of 0.5 mM dithiothreitol. For unfolding equilibrium, Mtb DsbE (final concentration of 7 µM) was dissolved in difference concentrations of GdnHCl and incubated for 3 h at 25 °C. Data were analyzed according to the two-state assumption (39, 40). The standard changes of folding free energy were calculated according to Equation 7.

(Eq. 7)
The difference in stability between the oxidized and reduced forms of DsbE protein was calculated as in Equation 8.

(Eq. 8)

Oxidase activity of Mtb DsbE: Refolding of Hirudin—Hirudo medicinalis hirudin (Sigma) refolding was performed as described (41). Reduced, unfolded hirudin (28 µM) was incubated with 84 µM oxidized Mtb DsbE in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA at 25 °C. At different time points, 100-µl samples were removed, and the reaction was quenched by the addition of 20 µl of formic acid and 20 µl of acetonitrile, respectively. The samples were than analyzed using a Phenomenex Primesphere 5-µm C18 column using a gradient from 30 to 37% D with 0.1% trifluoroacetic acid over 70 min at a flow rate of 1 ml/min. Buffer C was 0.1% trifluoroacetic acid in an aqueous solution, and buffer D was 0.1% trifluoroacetic acid in 80% acetonitrile. Peaks were detected using the Waters W474 fluorescence detector with 274-nm excitation and 325-nm emission wavelength settings.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Structure of Mtb DsbE—The crystal structure of Mtb DsbE consists of a main domain that contains a thioredoxin fold, with its distinct structural motif consisting of a fourstranded {beta}-sheet made up of {beta}3, {beta}4, {beta}6, and {beta}7 and three flanking {alpha}-helices corresponding to {alpha}3, {alpha}5, and {alpha}6 (Fig. 2a). In addition to the thioredoxin fold domain, a short 310-helix ({alpha}1), two {beta}-strands ({beta}1 and {beta}2), and another short 310-helix ({alpha}2) appear at the N terminus. A long {alpha}-helix ({alpha}4) and a {beta}-strand ({beta}5) (forming a five-stranded {beta}-sheet) are found after the {beta}3-{alpha}3-{beta}4 motif of the thioredoxin fold.



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  		FIG. 2.
Three-dimensional structure of Mtb DsbE and sequence comparisons of DsbE homologs. a, ribbon diagram of the monomer. {alpha}-Helices, 310-helices, and {beta}-strands are shown in red, purple, and blue, respectively. The active site Cys sulfur atoms and {beta}-carbon atoms are shown in yellow. This image was generated using RIBBONS. b, The Mtb DsbE active site; a view of the final 2Fo - Fc electron density map at the active site region. The electron density is contoured at 1.2{sigma}. The distance between the two Cys sulfur atoms is 3.69 Å. This image was generated using RIBBONS. c, sequence comparison of DsbE homologs. The first and second diagrams depict the B. japonicum DsbE monomer (blue; sequence also in blue) and the Mtb DsbE monomer (red; sequence also in red) in terms of secondary structure elements, respectively. Residues labeled with a number sign have two distinct conformations in the Mtb DsbE structure. Residues with a dollar sign are the residues that form a hydrogen bond between {alpha}-helix 3 and {beta}-strand 3. The sequence alignment of DsbE homologs was made with ClustalW. The DsbE proteins cluster into two main groups, Gram-negative bacteria and Gram-positive bacteria, with the extremophile (D. radiodurans) separating the two groups. The yellow boxes show blocks of conserved residues in the Gram-negative bacterium that are not seen in Gram-positive bacterium. MT, M. tuberculosis; MA, Mycobacterium avium; AO, Amycolatopsis orientalis; BS, Bacillus subtilis; SC, Streptomyces coelicolor; MS, Mycobacterium smegmatis; BH, Bacillus halodurans; DR, D. radiodurans; NM, Neisseria meningitides; RC, Rhodobacter capsulatum; PD, Paracoccus denitrificans; BJ, B. japonicum; PC, Pantoea citrea; EC, E. coli; VC, V. cholerae; PA, Pseudomonas aeruginosa; PM, Pasteurella multocida; HI, Hemophilus influenzae.

 
Active Site Dithiols—The two active site cysteines, Cys36 and Cys39, are reduced as determined by the 3.69 ± 0.30 Å distance between the two S{gamma} atoms (Fig. 2b). A disulfide bond linking the two residues would be expected to have a distance of 2.03 ± 0.25 Å between the two S{gamma} atoms (42). The cysteines adopt a right-handed hook confirmation at the N terminus of helix {alpha}3, which is consistent for the active site cysteines in the thioredoxin superfamily fold. Only the S{gamma} atom of Cys36 in the dithiol is exposed on the protein surface, whereas S{gamma} of Cys39 is buried, which is usual for a thioredoxin superfamily protein. The sulfur atom of Cys36 is stabilized by hydrogen bonds to the O{epsilon}1 atom of Gln100 (3.05 Å) and to the amide nitrogen atom of Pro37 (3.34 Å) and the amide nitrogen atom of Phe38 (3.35 Å); the S{gamma} atom of Cys39 is not in a hydrogen-bonding network but does have a hydrophobic interaction with conserved cis-Pro101 (4.15 Å) as does Cys36.

Crystallographic Dimeric Contacts—There is a crystallographic homodimeric interface that could possibly be in the transient heterodimer interface for oxidoreductase activity, especially since the interface is in the vicinity of the CXXC active site region. The surface area of each monomer is 6400 Å2, and the buried surface area of each monomer at the interface is 600 Å2, which is ~10% of the total surface area and corresponds to a transient homodimeric interface (43). A {pi}-cation interaction occurs between the aromatic ring of Trp35' and the active site, Cys36 (the prime on position 35' indicates that this residue is on the neighboring molecule). Arg64 from one monomer buries itself into the other monomer, forming hydrogen bonds with symmetry-related residues. The NH1 and NH2 atoms of Arg64 both hydrogen-bond to atom N{epsilon}1 of Trp99' and the oxygen atom of Asn117' respectively, via a water molecule, and both hydrogen-bond to the oxygen atom of Gln100'. Furthermore, the N{epsilon} atom of Arg64 interacts with the oxygen atom of Pro118'. Other notable homodimer interface hydrogen bonds are atom N{epsilon}1 of Trp99 to atoms O{epsilon}1 of Gln100' and to the oxygen atom of both Val97' and Pro98'. There are also hydrophobic interactions seen between the rings of Trp99 and Trp92'.

Structural Comparisons—The known structure with the highest structural similarity to Mtb DsbE is Bradyrhizobium japonicum CcmG/DsbE (B. japonicum DsbE) (44), a Gram-negative bacterial DsbE periplasmic protein. The alignment of the two structures gives a root mean square deviation over backbone atoms of 1.8 Å. The structure with the second highest similarity is B. japonicum TlpA (45), which is involved in the maturation of cytochrome aa3 (root mean square deviation of 2.1 Å). Fig. 3, a-c, shows the structural similarity between the three proteins, Mtb DsbE, B. japonicum DsbE, and B. japonicum TlpA, respectively. The most obvious difference between these three proteins is that the active site cysteines in the B. japonicum DsbE and B. japonicum TlpA structures are in their oxidized states but are in their reduced states in the Mtb DsbE structure.



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  		FIG. 3.
Ribbon diagrams of Mtb DsbE and two of the proteins with similar structures. a-c, ribbon diagrams of the structures with the active site cysteines indicated with an arrow. These images were generated using RIBBONS. a, Mtb DsbE; b, B. japonicum DsbE; c, B. japonicum TlpA. It should be noted that all three structures have similar topology, although in Mtb DsbE the active site is in its reduced form, whereas in B. japonicum DsbE and B. japonicum TlpA the active sites are in their oxidized form.

 
Gram-positive Mtb DsbE Protein Functions Differently from Gram-negative DsbE Proteins—The function of DsbE homologs in Gram-positive bacteria has not previously been characterized. A phylogenetic tree derived from prokaryotic homologs to E. coli DsbE shows that the Gram-positive and Gram-negative DsbE homolog proteins cluster into two groups with the extremophile Deinococcus radiodurans separating the two groups (data not shown). The divergence of Gram-negative and Grampositive DsbE homologs and the difference in redox state of Mtb DsbE and B. japonicum DsbE crystal structures suggest that Gram-positive DsbE and Gram-negative DsbE homologs may function differently.

Determination of the Redox Potential of Mtb DsbE—To further characterize Mtb DsbE, the redox potential relative to that of glutathione was determined, which compares the ability of reduced glutathione to transfer electrons to a protein. The Keq of Mtb DsbE is ~0.25 ± 0.2 mM (Fig. 4a). The corresponding standard redox potential (E'0) calculated for Mtb DsbE is -128 ± 12 mV. In comparison with the standard redox potential for E. coli DsbA (-124 mV) (46), which is an oxidant, and E. coli thioredoxin (-269 mV) (35), which is a reductant, the standard redox potential for Mtb DsbE (-128 mV) suggests that DsbE is an oxidant. In contrast, the standard redox potentials for Gram-negative DsbE proteins (-217 to -175 mV) (47-49) correspond to these proteins being weak reductants. Since Mtb DsbE is an oxidant and Gram-negative DsbE proteins are weak reductants, this reinforces the hypothesis that Gram-negative and Gram-positive DsbE proteins function differently.



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  		FIG. 4.
Biochemical Characterization of Mtb DsbE. a, redox equilibrium of Mtb DsbE with glutathione. The y axis represents the proportion of Mtb DsbE present in the reduced form at equilibrium with various mixtures of reduced (GSH) and oxidized (GSSG) glutathione (x axis), which was measured by exploiting the difference in fluorescence (347 nm) of reduced Mtb DsbE compared with oxidized Mtb DsbE. Equilibrium concentrations of oxidized and reduced Mtb DsbE and GSH and GSSG were calculated as described (33, 34). The equilibrium constant was calculated to be 0.25 ± 0.2 mM, which is consistent with Mtb DsbE acting as an oxidant. b, determination of the pKa values of the active site cysteine, Cys36, in Mtb DsbE. The absorption specific to reduced Mtb DsbE protein is shown as a function of pH (x axis) compared with the fraction of Cys36 thiolate (y axis). The pKa value of Cys36 was calculated by fitting the data points as described in Ref. 9. The pKa of Cys36 in Mtb DsbE is 5.0 ± 0.2. c, GdnHCl-dependent unfolding/folding of Mtb DsbE. GdnHCl-dependent unfolding was monitored by circular dichroism spectroscopy at 222 nm of oxidized Mtb DsbE (shown as diamonds) and reduced Mtb DsbE (shown as triangles). Refolding of oxidized Mtb DsbE is shown by open diamonds, and refolding of reduced Mtb DsbE is shown by open triangles. GdnHCl concentration is the x axis, and the y axis represents the fraction of native (folded) protein. Since the reduced form of Mtb DsbE unfolds at higher concentrations of GdnHCl than the oxidized form, the reduced form of Mtb DsbE is its more stable form. d, analysis of the refolding kinetics of reduced and unfolded hirudin. A 3-fold molar excess of oxidized Mtb DsbE was used and incubated with reduced unfolded hirudin at pH 7.0 and 25 °C. Samples were removed as indicated on each time course and quenched as described. The peak for the oxidized hirudin is marked by an asterisk. O and R mark the retention time for oxidized and reduced hirudin, respectively. The rightmost trace is after a 24-h incubation in the absence of Mtb DsbE (w/o DsbE).

 
To confirm the redox potential that was determined by equilibrium incubation using GSH/GSSG as a reference, we applied a method based on the analysis of the direct protein-protein redox equilibrium between Mtb DsbE and the protein E. coli DsbC, which has a well established redox potential of E'0 is -135 mV (37). By analyzing the equilibrium between E. coli DsbC and Mtb DsbE, we determined the redox potential of Mtb DsbE (E'0) to be -128 ± 3 mV. This is in very good accordance with the value determined using GSH/GSSG as a reference. This makes Mtb DsbE one of the most oxidizing proteins known.

Determination of the pKa Value of Mtb DsbE—Determination of the pKa value of the Mtb DsbE solvent-exposed active site cysteine provides further evidence that Mtb DsbE is functionally divergent from Gram-negative DsbE proteins. The pKa value of the active site cysteine of Mtb DsbE (Cys36) was measured by observing the change in absorption of the cysteines at 240 nm over a pH range of pH 2-9 (Fig. 4b). The pKa value of the solvent-exposed active site cysteine (Cys36) is 5.0 ± 0.2. This is relatively acidic compared with the solvent-exposed active site cysteine of E. coli thioredoxin (pKa of 7.5) (50) and E. coli {Delta}57DsbE (pKa of 6.8) (47) and that of reduced glutathione (pKa of 8.7), although not as acidic as E. coli DsbA, which is a known oxidant whose pKa of the solvent-exposed active site cysteine is 3.5 (51). The acidic nature of the solvent-exposed Cys36 provides further evidence that Mtb DsbE has different biochemical characteristics than its Gram-negative DsbE homologs.

Thermodynamic Properties of the Redox Forms—To compare stabilities of the different redox forms of Mtb DsbE, guanidine hydrochloride-induced unfolding and refolding of both oxidized and reduced forms was examined by circular dichroism. The reduced form of Mtb DsbE is more stable than that of the oxidized form, given that the reduced form of the protein denatures at a higher concentration of guanidine hydrochloride than the oxidized form (Fig. 4c). Calculation of the free energy change ({Delta}{Delta}Gredox) between the reduced and oxidized form of Mtb DsbE suggests that the reduced form is 12.4 ± 4 kJ/mol more stable than the oxidized form. E. coli DsbA (an oxidant) is also more stable in its reduced form, with a {Delta}{Delta}Gredox of 16.3 ± 3.6 kJ/mol (41), whereas E. coli thioredoxin (a reductant) is 16.3 ± 2.4 kJ/mol more stable in its oxidized form (52). Thus, the increased stability of Mtb DsbE in its reduced form is consistent with Mtb DsbE being energetically more stable as an oxidant.

Oxidase Activity of Mtb DsbE and Hirudin Refolding—In order to investigate the oxidative protein folding ability of Mtb DsbE, we tested its ability to oxidize hirudin from H. medicinalis. Hirudin is a 6.9-kDa protein that functions as an inhibitor of thrombin. It contains three intramolecular disulfide bridges that connect residues 6-14, 16-28, and 22-39. The regeneration of native recombinant hirudin from the reduced unfolded form to the fully oxidized native state was carried out in the presence and absence of Mtb DsbE in stoichiometric quantities. In the absence of added Mtb DsbE, a small quantity of spontaneous (presumably air-mediated) oxidation of hirudin occurred to generate randomly oxidized hirudin, as has previously been observed, but negligible native hirudin was generated (Fig. 4d). In contrast, Mtb DsbE was able to oxidize hirudin from a denatured reduced state to a completely folded and oxidized state (Fig. 4d). This shows that Mtb DsbE is capable of oxidizing substrate proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Comparison of Mtb DsbE with Gram-negative DsbE Homologs—Several lines of evidence imply that Mtb DsbE does not function in the same way as its Gram-negative homologs. DsbE proteins in Gram-negative bacteria have been shown to be weak reductants, whose solvent-exposed active site cysteines have pKa values around 6.5-6.8. We have shown that Mtb DsbE is an oxidant, and its solvent-exposed, active site cysteine has a lower pKa (5.0) than those of Gram-negative bacterial DsbE homologs. These differences and the observation that the sequences of Gram-positive DsbE homologs cluster together imply that Gram-positive DsbE proteins, and in particular Mtb DsbE, have a different biochemical function than Gram-negative DsbE homologs.

Sequence and structural alignments of Gram-negative and Gram-positive DsbE homologs also suggest different functions. They show two regions that are conserved among Gram-negative DsbE homologs but are significantly different in Gram-positive DsbE proteins (Fig. 2c, yellow boxes). The first N-terminal region, conserved in the Gram-negative DsbE homologs contains an insertion of 15 residues that are absent in the Mtb DsbE structure (Fig. 2c). The additional 15 residues, together with alterations in surrounding residues, generate a region that is structurally similar to a {beta}-hairpin. This region forms a distinct groove on the protein's molecular surface (Fig. 5b), which has been shown to have the required architecture for interaction with the protein partners of B. japonicum DsbE (44). Sequence alignments show that this region is well conserved among Gram-negative DsbE proteins (Fig. 2c, N-terminal yellow box). In contrast, the Mtb DsbE has a shorter N-terminal region, the 310-{beta}-{beta}-310 segment (Fig. 2c), which does not form a groove on the protein's molecular surface (Fig. 5a). This region is poorly conserved among Gram-positive DsbE proteins. Since this groove is thought to play an important role in protein-protein interactions of B. japonicum DsbE, we may conjecture that the protein interaction partners of Mtb DsbE differ from that of B. japonicum DsbE (44), that Mtb DsbE does not use this region for protein-protein interactions, or both. A second region of conserved residues in Gram-negative DsbE homologs contains the Gram-negative DsbE protein motif (53), 152GVXGXPET159, which is located in a solvent-exposed loop region between {alpha}3 and {beta}4 in the B. japonicum DsbE structure (Fig. 2c, second yellow box). The side chains of these residues pack tightly around the protein backbone and do not protrude into the solvent. In Gram-positive bacteria, this motif is not conserved, and the corresponding sequence in Mtb DsbE, 96NVPWQPAF103, contains residues that protrude from the protein backbone to form a potential homodimer interface. The B. japonicum DsbE crystal structure contains no crystallo-graphic or potential homodimer interface (44). In summary, the differences in the sequence conservation between the Gram-negative DsbE proteins and Gram-positive DsbE proteins reinforce our conclusion that Mtb DsbE functions differently from Gram-negative DsbE proteins.



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  		FIG. 5.
Molecular surface representation of monomeric and active site of Mtb DsbE and B. japonicum DsbE. a, illustration of the transparent molecular surface of Mtb DsbE with the ribbon diagram of the structure in orange. b, illustration of the transparent molecular surface of B. japonicum DsbE with the ribbon diagram of the structure in purple. The potential protein interaction groove is indicated. Helix {alpha}3 is labeled in both structures. The figure shows that there is no potential protein interaction groove seen in the Mtb DsbE structure as compared with the B. japonicum DsbE structure. c and d, ribbon diagrams of the active sites of Mtb and B. japonicum DsbE structures. The {alpha}-helices and {beta}-strands are shown in cyan and green, respectively. The active site cysteines and amino acid pair atoms are shown in green, red, blue, and yellow, representing carbon, oxygen, nitrogen, and sulfur, respectively. These images were generated using RIBBONS. c, Mtb DsbE; d, B. japonicum DsbE. The hydrogen bond between Trp30 and Glu42 in the Mtb DsbE structure maintains a conformation in which the active cysteines are in their reduced form. In contrast, the hydrogen bond between Asn86 and Glu98 in the B. japonicum DsbE structure maintains a conformation that allows a disulfide bond to form between the active site cysteines.

 
Comparison of the active sites of B. japonicum DsbE and Mtb DsbE structures is also consistent with the hypothesis that Gram-negative and Gram-positive homologs of DsbE function differently. B. japonicum DsbE crystallized in its oxidized form, and Mtb DsbE crystallized in its reduced form (Fig. 3, b and a, respectively), suggesting that the active site environment differs between the two proteins. The active site of B. japonicum DsbE is unusually acidic, due to residues Asp97, Glu98, and Glu158 (which are conserved among the Gram-negative DsbE proteins), which may account for the reducing power of B. japonicum DsbE compared with Mtb DsbE. The corresponding residues in Mtb DsbE (Ala41, Glu42, and Ala102) are not acidic except for Glu42, which is conserved throughout the DsbE homologs (Fig. 2c). Alignment of the two structures in the vicinity of the active site loop (residues Trp30-Glu42 and Asn86-Glu98 in the Mtb DsbE and B. japonicum DsbE, respectively) gives a root mean square deviation over backbone atoms of 0.42 Å (Fig. 5, c and d). Thus, the redox state of DsbE proteins is not dependent on a conformational change near the active site, implying that the redox state of the protein is not determined by the architecture of the active site.

Structural analysis implicates an amino acid pair that contributes to the stability of the reduced and oxidized forms of the Dsb proteins. In the reduced form of Mtb DsbE, the amino acid pair Trp30 and Glu42 are flanking the active site residues in {beta}-strand 3 ({beta}3) and {alpha}-helix 3 ({alpha}3), respectively (Fig. 5c), and form hydrogen bonds between N{epsilon}1 of Trp30 and both O{epsilon}1 and O{epsilon}2 of Glu42 (2.90 and 2.77 Å). This interaction probably contributes to the stability of the active site loop to form a conformation where the reduced thiol form of the active site cysteines is favored, and therefore Mtb DsbE is an oxidant. This amino acid pair is well conserved throughout the Gram-positive DsbE proteins. In comparison, within the B. japonicum DsbE structure (44), which crystallized in its oxidized form, the corresponding residues are Asn86 and Glu98. These two residues also form a hydrogen bond (3.08 Å) across the {beta}-strand and {alpha}-helix (Fig. 5d), possibly maintaining the active site loop in a conformation that favors the disulfide form of the active site cysteines. In all of the Dsb and thioredoxin structures determined thus far, there is a corresponding conserved amino acid pair that forms a hydrogen bond between the {beta}-strand and {alpha}-helix containing the active site cysteines in the protein's most stable form. For example, the structure of E. coli DsbA, an oxidant, shows the amino acid pair to be Glu37 and Lys58. In the reduced form of DsbA (the more stable form), the hydrogen bond between Glu37 and Lys58 has a distance of 2.92 Å, whereas in the oxidized form, the distance is ~0.75 Å greater (54). This implies that the hydrogen bonding flanking the active site in thioredoxin-like proteins may influence their redox state by favoring conformations in which active site cysteines are most stable in their reduced or oxidized states.

Biological Implications—In Gram-negative bacteria, it has been proposed that DsbE proteins are involved in the maturation of cytochrome c. Cytochrome c maturation converts a linear polypeptide, the apocytochrome, into a three-dimensional structure that contains one or more convalently bound, redoxactive heme co-factors. There are at least three systems of cytochrome c maturation of varying complexity (55, 56). Gram-negative bacteria cytochrome c maturation utilizes a well characterized pathway, System I, which contains periplasmic anchored DsbE (6, 56). Since Gram-negative DsbE has been proposed to play a role as a reductant, and we have shown that Mtb DsbE is an oxidant, the reduction of the cysteines of apocytochrome c would be an unfavorable reaction. This suggests that Mtb DsbE is not involved in cytochrome c maturation via System I. Cytochrome c maturation in Gram-positive bacteria is thought to utilize System II, which is a less well characterized pathway as compared with System I (55, 57). System II also contains a predicted thioredoxin-like protein (Ccs1/ResB), and the M. tuberculosis genome contains all of the known genes in System II cytochrome c maturation. Thus Mtb DsbE may be involved in cytochrome c maturation by the System II pathway.

An alternative role for Mtb DsbE could be to function as a disulfide bond-forming (Dsb) protein. The M. tuberculosis genome contains no genes that encode for other homologs of Dsb proteins, such as E. coli DsbA, DsbC, or DsbG. In Gram-negative bacteria, Dsb proteins function in the periplasm. Because M. tuberculosis is a Gram-positive bacterium and does not contain a periplasmic space, Mtb DsbE might function extracellularly within the cell wall environment. If so, Mtb DsbE might function as a disulfide isomerase to ensure that secreted or surface-associated proteins have correctly formed disulfide bonds. It has been predicted that greater than 60% of the 161 predicted secreted proteins of M. tuberculosis contain at least one disulfide bond.2 However, in vitro, we found no activity for disulfide bond isomerase in Mtb DsbE (data not shown); nor did previous studies (24). Therefore, if Mtb DsbE functions as an isomerase, this suggests a high specificity of Mtb DsbE for its functional protein partners.

The final option and the one that we favor is that Mtb DsbE may have a similar function to E. coli DsbA, which catalyzes the oxidation of reduced, unfolded proteins with disulfide bonds (10, 11). This hypothesis is supported by the observation that Mtb DsbE and E. coli DsbA are both oxidants, and their solvent-exposed active site cysteines both have relatively low pKa values, 5.0 and 3.4 (38), respectively. In addition, Mtb DsbE has been shown to have oxidase activity as it reoxidizes reduced hirdurin. In fact, the NCBI conserved domain site selects the first 50 residues of Mtb DsbE in having a DsbA domain. Therefore, Mtb DsbE may catalyze the oxidation and folding of reduced, unfolded proteins as they are secreted into the extracellular space of the M. tuberculosis cell wall environment.

In summary, structural and functional analysis of Mtb DsbE suggests that it has a similar function to E. coli DsbA, which catalyzes the oxidation of reduced, unfolded secreted proteins to form disulfide bonds (10, 11). Hence, Mtb DsbE may be involved in virulence, since many secreted proteins may depend on the oxidase activity of Mtb DsbE to form correctly folded proteins. Interestingly, although the overall structures of Gram-negative and Gram-positive DsbE proteins have similar thioredoxin-like folds, the structural differences between these two proteins imply that they function differently (the N-terminal regions, the potential interaction interfaces, and the redox state of the active sites differ between the two proteins). Biochemical analysis of Mtb DsbE confirms this assumption, since Mtb DsbE is an oxidant probably acting upon secreted proteins, whereas B. japonicum DsbE is a weak reductant acting upon apocytochrome c. Thus, the determination and analysis of the structure of Mtb DsbE along with comparison with homologous sequences and structures has provided an opportunity to make specific predictions about protein function that were confirmed biochemically.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1LU4 [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by Grant NIH 20298-001-01 from the National Institutes of Health, Department of Energy-BER, and Howard Hughes Medical Institute (to D. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by Deutsche Akademie der Naturforscher Leopoldina Grant BMBF-LPD 9901/8-79. Back

** Supported by National Institutes of Health Grant GM646222. Back

§§ To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Institute and UCLA-DOE Institute of Genomics and Proteomics, P.O. Box 951570, Los Angeles, CA 90095-1570. E-mail: david{at}mbi.ucla.edu.

1 The abbreviations used are: Dsb, disulfide bond-forming proteins; Mtb, M. tuberculosis; Rv number, Sanger center notation for each gene in Mtb. Back

2 P. Mallick, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Michael Sawaya and Duilio Cascio for invaluable help with data collection and general crystallography and Parag Mallick for statistics on disulfide bonds in the M. tuberculosis genome. We also thank Brookhaven National Laboratory for the use of beamline X8C of the National Synchrotron Light Source, particularly Joel Berendzen, Li Wei Hung, and Leonid Flaks.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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