Structural and biochemical characterization of the essential DsbA-like disulfide bond forming protein from Mycobacterium tuberculosis
© Chim et al.; licensee BioMed Central Ltd. 2013
Received: 9 July 2013
Accepted: 11 October 2013
Published: 18 October 2013
Bacterial D is ulfide b ond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Previously, we have shown that Mycobacterium tuberculosis Mt-DsbE and Mt-DsbF aid in vitro oxidative folding of proteins. The M. tuberculosis proteome contains another predicted membrane-tethered Dsb protein, Mt-DsbA, which is encoded by an essential gene.
Herein, we present structural and biochemical analyses of Mt-DsbA. The X-ray crystal structure of Mt-DsbA reveals a two-domain structure, comprising a canonical thioredoxin domain with the conserved CXXC active site cysteines in their reduced form, and an inserted α-helical domain containing a structural disulfide bond. The overall fold of Mt-DsbA resembles that of other DsbA-like proteins and not Mt-DsbE or Mt-DsbF. Biochemical characterization demonstrates that, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced, denatured hirudin. Moreover, on the substrates tested in this study, Mt-DsbA has disulfide bond isomerase activity contrary to Mt-DsbE and Mt-DsbF.
These results suggest that Mt-DsbA acts upon a distinct subset of substrates as compared to Mt-DsbE and Mt-DsbF. One could speculate that Mt-DsbE and Mt-DsbF are functionally redundant whereas Mt-DsbA is not, offering an explanation for the essentiality of Mt-DsbA in M. tuberculosis.
KeywordsMycobacterium tuberculosis Disulfide bond X-ray crystallography DsbA Vitamin K epoxide reductase Oxidoreductase
Correct folding and disulfide bond formation is essential for the function of many secreted proteins including bacterial toxins, and their formation is facilitated by d is ulfide b ond forming (Dsb) oxidoreductase proteins, which usually contain a conserved thioredoxin (TRX) fold . Protein disulfide bonds can serve structural roles, and thus are often buried in the core of a protein. However, in the case of Dsb proteins, partially exposed disulfide bonds in the TRX-fold CXXC motif have catalytic roles in protein folding, electron transport and bioenergetics in a variety of organisms [2, 3].
The Dsb proteins of Escherichia coli are the best characterized, and reside in its periplasm to correctly fold disulfide bond containing secreted and cell-wall proteins . E. coli DsbA (Ec-DsbA) catalyzes the oxidation of disulfide bonds in reduced, unfolded proteins [5, 6], and is then re-oxidized by ubiquinone via E. coli DsbB (Ec-DsbB), an inner membrane transmembrane protein, which in turn is oxidized by the electron transport pathway [7, 8]. E. coli DsbC (Ec-DsbC) and E. coli DsbG (Ec-DsbG) serve as proofreading disulfide isomerases that are able to break and correctly reform non-native protein disulfide bonds, thus ensuring that these important secreted proteins are functionally active [9, 10]. E. coli DsbD (Ec-DsbD) is responsible for maintaining Ec-DsbC and Ec-DsbG in their active redox states and is a transmembrane protein also spanning the inner membrane . Finally, E. coli DsbE (Ec-DsbE) is a reductant involved in cytochrome c maturation , its redox partner is also proposed to be Ec-DsbD . Dsb-like homologs have been found in many prokaryotes , including gram-positive bacteria where they also appear to be widespread. As gram-positive bacteria have no spatially defined periplasmic compartment, the precise function and membrane bound redox partners of these Dsb-like proteins appear to differ from those of gram-negative bacteria .
Dsb proteins, and in particularly DsbA, have been shown to be involved in virulence of toxin-secreting gram-negative bacteria such as Yersinia pestis , Shigella sp. , Vibrio cholerae [17, 18] and E. coli . Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium responsible for tuberculosis (TB), which causes approximately 1.4 million deaths and 8 million new cases per year . Mtb secreted proteins have many different functions including those associated with virulence, pathogenicity and cell-wall maintenance. Within the Mtb proteome, it has been predicted that over 180 proteins are secreted, of which ~ 60% may contain disulfide bonds based on their cysteine content, thus suggesting that Dsb proteins may play an important role in the correct folding of secreted proteins . Mtb EspA is one such secreted protein that may require the folding assistance of Mtb Dsb proteins. The single disulfide bond within Mtb EspA has been found to have an important role in disease progression in mice as well as maintaining cell wall integrity , highlighting a crucial link between disulfide bond formation and virulence in Mtb. One could speculate that interruption of the Mtb Dsb-assisted folding pathways may prevent mycobacterial infectivity and viability. Therefore, the study of Mtb Dsb protein systems may offer new insight into its virulence and may provide novel anti-TB drug targets.
In this study, we have determined the 1.9 Å-resolution structure of the soluble form of Mt-DsbA (residues 46–255). The overall fold of Mt-DsbA is reminiscent of Ec-DsbA  and consists of two domains, a TRX domain and an inserted α-helical domain. The Mt-DsbA TRX domain active site CXXC motif cysteines are reduced while a stabilizing disulfide bond is observed in the α-helical domain [35, 36]. Unlike Mt-DsbE and Mt-DsbF, Mt-DsbA does not have the ability to catalyze the oxidative folding of hirudin [21, 25]. However Mt-DsbA possesses disulfide bond isomerase activity as confirmed by its ability to catalyze the refolding of scrambled ribonuclease A (scRNaseA) whereas Mt-DsbE and Mt-DsbF do not. This study represents the structural and functional characterization of an essential Mtb Dsb protein, Mt-DsbA, and suggests that Mt-DsbA likely acts on a distinct protein substrate set as compared to Mt-DsbE and Mt-DsbF due to their functional differences.
The structure of Mt-DsbA
Mt-DsbA is predicted to be either secreted into the periplasm by a signal peptide (SignalP)  or tethered to the inner membrane by an N-terminal transmembrane helix (TMHMM) . A recent paper suggested that Mt-DsbA is a membrane-tethered protein , prompting the investigation of the soluble form of Mt-DsbA encoding residues 46–255, as suggested by TMHMM.
Mt-DsbA has structural similarity to other bacterial DsbA proteins
Mt-DsbA cannot oxidatively fold reduced and denatured hirudin
Mt-DsbA does not have the ability to reduce insulin
Insulin contains two polypeptide chains (A and B) and has one intramolecular and two intermolecular disulfide bonds. Reduction of these disulfide bonds results in the dissociation of chains A and B, where chain B is insoluble and aggregates. Thus, the reduction of insulin may be assessed by following the increase in turbidity at 650 nm, which is due to the aggregation of chain B and can be determined in the presence and absence of a disulfide reductase. We determined the rate of insulin reduction in the presence of either Mt-DsbA, Mt-DsbE, Mt-DsbF or positive controls (Ec-DsbA and Ec-DsbC). Ec-DsbC and Ec-DsbA both possess insulin reductase activity; however Ec-DsbC is a stronger reductase than Ec-DsbA (Figure 4B). In contrast, in the presence of Mt-DsbA, Mt-DsbE and Mt-DsbF insulin exhibited basal-levels of aggregation similar to that of insulin in the absence of Dsb protein, suggesting that these Dsb proteins are unable to reduce insulin under the conditions tested (Figure 4B).
Mt-DsbA is able to refold scrambled RNaseA
Mt-DsbA contains some residues characteristic of the E. coli disulfide bond isomerases Ec-DsbC and Ec-DsbG (Asp in the DXXCXYC motif, Thr in cis-Pro loop). To determine whether Mt-DsbA has isomerase activity, we tested Mt-DsbA catalyzed recovery of active RNaseA from oxidized, disulfide-scrambled RNaseA (scRNaseA). This assay revealed that Mt-DsbA possesses scRNaseA isomerase activity and the active site CXXC cysteines are required for activity, as Cys89Ser and Cys92Ser mutations render Mt-DsbA inactive (Figure 4C). However, the structural disulfide bond in the inserted α-helical domain does not play a role in this activity as the Cys140Ser and Cys192Ser mutants retain similar isomerase activity to wild-type Mt-DsbA (Figure 4C). Of note, Wp-DsbA also contains a structural disulfide bond in its α-helical domain (Figure 3D), and mutation of these cysteines to alanines does not affect its disulfide bond isomerase activity , as observed for Mt-DsbA. Moreover, neither Mtb-DsbE nor Mt-DsbF can catalyze the refolding of scRNaseA to produce active RNaseA, and thus do not appear to have isomerase activity under the conditions tested (Figure 4C). These results demonstrate that Mt-DsbA has protein disulfide isomerase activity, while Mt-DsbE and Mt-DsbF do not possess this activity on the substrates tested in this study.
Comparisons of molecular surfaces and functions of DsbA-like proteins to Mt-DsbA
Ec-DsbA can catalyze the reduction of insulin in the presence of DTT ; in contrast, Mt-DsbA does not show such activity, which may be a consequence of its less hydrophobic patch above the CXXC active site motif (Figures 5A & B). Previous studies of Sa-DsbA demonstrate that, like Mt-DsbA, it has no ability to catalyze the reduction of insulin , and both Mt-DsbA and Sa-DsbA have a similar negatively charged molecular surface above the CXXC motif (Figures 5A & C). However, when the preceding cis- Pro Sa-DsbA residue, Thr153 was mutated to Val as observed in the Ec-DsbA cis-Pro loop (Figures 3G, 6B & C), some insulin reduction activity was restored while Sa-DsbA isomerase activity remained unchanged. In the Wp-DsbA study , a similar Thr to Val mutation in the cis-Pro motif (Figure 6D) also demonstrated more insulin reductase activity together with reduced isomerase activity compared to wild-type Wp-DsbA, and the Wp-DsbA Thr172Val mutant has a similar activity profile as Ec-DsbA . These observations suggest that the residue preceding cis-Pro influences substrate recognition and, in part the activities of Dsb proteins, leading to the speculation that lack of insulin reduction activity for Mt-DsbA may also result from disruption of substrate binding at the negatively charged patch (Figure 5A).
Structural and functional comparison of Mt-DsbA to Mt-DsbE and Mt-DsbF
Besides structural differences, Mt-DsbA exhibits functional differences compared to Mt-DsbF and Mt-DsbE. Both Mt-DsbE and Mt-DsbF can oxidatively fold reduced and denatured hirudin [21, 25] whereas Mt-DsbA does not possess this activity. In contrast, only Mt-DsbA exhibits isomerase/oxidizing activity on the substrate, scRNaseA. These results suggest two features that differentiate Mt-DsbA from Mt-DsbE and Mt-DsbF. First, Mt-DsbA probably functions on a separate subset of Mtb substrates compared to Mt-DsbE and Mt-DsbF. Second, one could postulate that Mt-DsbE and Mt-DsbF have redundant activities even though they have negatively correlated gene expression profiles , whereas Mt-DsbA is the sole protein to carry out its unique function in the Mtb Dsb system (Figure 1), and thus Rv2969c is an essential gene .
Implications for Mt-DsbA in host-pathogen interactions
A previous study demonstrated that Mt-DsbA localized on the Mtb membrane in vitro, and that it also contains several host-cell binding regions . A twenty amino acid Mt-DsbA peptide was used to produce antibodies that demonstrated that Mt-DsbA is exposed on the mycobacterial cell surface. This peptide includes α-helix (α3) within the inserted α-helical region (Figure 8B) and contains many charged residues exposed on the molecular surface of Mt-DsbA. Additionally, it was shown that two other twenty amino acid peptides of Mt-DsbA, one from the N-terminal region (including the first β-strand, β1) and the other from the C-terminal region (including the final α-helix, α8), Figure 2A, have the ability to bind to epithelial cells and inhibit Mtb invasion of these cells in a dose-dependent manner at high micromolar concentrations.
In this study, we show that Mt-DsbA is structurally distinct from Mt-DsbE and Mt-DsbF. Additionally, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced and denatured hirudin but can catalyze the refolding of scRNaseA. Taken together, these results imply that Mt-DsbA functions on a disparate set of substrates compared to either Mt-DsbE or Mt-DsbF. Furthermore, the knowledge that Mt-DsbA possibly facilitates mycobacterial interaction with host cells  implicates Mt-DsbA as a potential vaccine candidate. Moreover, as both Mt-DsbA and Mt-VKOR are encoded by essential genes , interruption of the Mt-DsbA and Mt-VKOR protein-protein interaction and its redox cycle, may greatly inhibit mycobacterial growth and virulence. Thus further investigation into host protein/Mt-DsbA, small molecule/Mt-DsbA and Mtb protein/Mt-DsbA interactions is warranted, as Mt-DsbA could be an excellent target for novel anti-TB therapeutics.
Cloning and mutagenesis
Primers used to produce the Mt-DsbA construct (residues 46–255)
5’ CCCATATGCGCGACGACAAGAAGGACGGCGTCGCGGG 3’
5’ CCAAGCTTGGATGTCGCGGTAGCAGCGGCCGAGTC 3’
Primers used for site-directed mutagenesis to produce Cys to Ser mutants
For: 5’ CTTCTACGAGGATTTCCTGTCTCCGGCGTGCGGCATATTC 3’
Rev: 5’ GAATATGCCGCACGCCGGAGACAGGAAATCCTCGTAGAAG 3’
For: 5’ GAGGATTTCCTGTGTCCGGCGTCCGGCATATTCGAGCGCGGTTCGG 3’
Rev: 5’ CCGAACCGCGCTCGAATATGCAGGCCGCCGGACACAGGAAATCCTC 3’
For: 5’ CTGCTGCGGCTTATTCCGTTGCCGACGAATC 3’
Rev: 5’ GATTCGTCGGCAACGGAATAAGCCGCAGCAG 3’
For: 5’ CAAGGTGCCCGACTCCATCAACAGCGGCAAG 3’
Rev: 5’ CTTGCCGCTGTTGATGGAGTCGGGCACCTTG 3’
Protein expression and purification
The mature form of Mt-DsbA residues (46–255) with a C-terminal HisTag was overexpressed from a pET30 plasmid containing a truncated Rv2969c gene using E. coli BL21(DE3)-Gold cells. Cells were grown at 37° C in LB medium containing 50 μg/ml of kanamycin. Protein expression was induced by adding 1 mM IPTG at an OD600 ~ 0.8 and grown for 4 h before harvesting. Cells were pelleted at 5,000 rpm for 10 min and then resuspended in wash buffer (50 mM Tris–HCl pH 7.4, 350 mM NaCl, 10 mM imidazole and 10% glycerol) containing phenylmethylsulfonyl fluoride and hen egg lysozyme, and then were lysed by sonication and centrifuged at 13,000 rpm for 40 min followed by filtration (0.22 μm) to remove cell debris before purification. The cell lysate was loaded on to a Ni2+-charged HiTrap column (5 mL) and washed with wash buffer before protein was eluted with a 10–500 mM linear imidazole gradient (100 ml) in which purified Mt-DsbA eluted between 200 and 300 mM imidazole. The fractions containing pure Mt-DsbA were collected and concentrated (Amicon, 10 kDa molecular mass cutoff) and then further purified by gel filtration on a Superdex 200 column (GE Healthcare) equilibrated with 20 mM Tris–HCl (pH 7.4), 150 mM NaCl using an AKTA FPLC. The selenomethionine-derivatized (SeMet)-Mt-DsbA was grown in M9 minimal medium supplemented with amino acids supplements (leucine, isoleucine, valine, 50 mg/L; phenylalanine, lysine, threonine, 100 mg/L; and selenomethionine 75 mg/L) adapted from a previously described protocol . The SeMet-Mt-DsbA and cysteine to serine mutants were purified as described for native Mt-DsbA.
Crystallization, data collection and structure determination
X-ray diffraction data collection and atomic refinement statistics for Mt-DsbA in its reduced form
P2 1 2 1 2
No of monomers per AS unit
Unit cell dimensions (Å)
71.0 × 76.7 × 86.9
pH of crystallization condition
Resolution range (Å)
Unique reflections (total)
Resolution range (Å)
No. of reflections (working/free)
Residues of Mt-DsbA
Chain A 56-255
Chain B 49-255
No. of protein atoms
No. of water molecules
R work/R free b, %
Most favorable region (%)
Additional allowed region (%)
Disallowed region (%)
PDB ID code
Oxidation and reduction of Mt-DsbA
To oxidize Mt-DsbA, 50 mM of oxidized glutathione (GSSG) was added to as-isolated Mt-DsbA in 50 mM Tris–HCl, 150 mM NaCl and incubated for 1 hour at room temperature. Oxidized Mt-DsbA was recovered by gel filtration in 50 mM Tris–HCl pH 7.4, 150 mM NaCl. To reduce Mt-DsbA, 100 mM dithiothreitol (DTT) was added to Mt-DsbA and incubated at 4°C overnight. Reduced Mt-DsbA was recovered by gel filtration in 50 mM Tris–HCl, 150 mM NaCl.
The redox state of the thiols was confirmed by the Ellman’s assay, which exploits the colorimetric change at 412 nm when 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is converted to 2-nitro-5-thiobenzoate upon cleavage of the disulfide bond by free thiols. This reaction is stoichiometric, thus allowing for accurate quantification of free thiols.
To test for Dsb-catalyzed oxidative folding of hirudin
To test for Mt-DsbA catalyzed oxidative folding of reduced, denatured hirudin in vitro, the experiment was carried out as previously described . Commercial H. medicinalis hirudin (Sigma) was purified to remove contaminants by reverse-phase HPLC on a Synergi 4 mm Hydro-RP column (250 × 4.6 mm, Phenomenex) at a flow rate of 0.5 ml/min. Solvents A and B used for reverse-phase HPLC were 0.1% trifluoroacetic acid in water and acetonitrile, respectively. Purified hirudin was reduced and denatured by incubation with 100 mM DTT, 6 M Gdn-HCl overnight and eventually desalted by ZipTip C4 (Millipore). To test for oxidative folding of reduced, denatured hirudin, 3 molar equivalents of oxidized Mt-DsbA (5 pmol) in 100 mM ammonium bicarbonate, pH 8.0 at 25°C and added to reduced, denatured hirudin (~1.5 pmol). Each 10 μl reaction was quenched at different time points by a 10-minute incubation at 50°C with 50 μl 6 M Gdn-HCl and followed by the addition of 0.5 μl 100 mM iodoacetamide for 15 minutes at 25°C. The samples were then desalted by ZipTip C4 (Millipore) and analyzed by MALDI-TOF mass spectrometry (Voyager). Experiments were repeated with no protein and with approximately three molar equivalents (5 pmol) of Mt-DsbF and Ec-DsbA as positive controls. The appearance of native hirudin (m/z 6765) is represented as a percentage of the total intensities of native and carbamidomethylated hirudin .
To test for Dsb-catalyzed reduction of insulin
To test for protein disulfide reductase activity of Mt-DsbA, experiments were carried out in vitro using the insulin reduction assay in the presence of DTT . 10 μM of Ec-DsbA, Ec-DsbC (as positive controls, Sigma) or Mt-DsbA in buffer containing 0.1 M phosphate buffer, pH 7.2, 2 mM EDTA and 0.33 mM DTT were mixed with 0.131 mM insulin to initiate the reaction. Insulin comprises A and B polypeptide chains connected by two disulfide bonds. Reduction of the disulfide bonds leads to precipitation of the insoluble B chain, and is followed spectroscopically as an increase in optical density at 650 nm. The solutions were monitored at 30 s intervals over a period of 80 min.
To test for Dsb-catalyzed refolding of scrambled RNaseA
In vitro isomerase activity of Dsb proteins were assessed utilizing scRNaseA as previously described . ScRNaseA was produced by first incubating native RNaseA (Sigma) in 50 mM Tris–HCl, pH 8.0 with 6 M Gdn-HCl and 0.1 M DTT overnight at room temperature. The reduced, unfolded RNaseA was acidified with 100 mM acetic acid, pH 4 and purified over a desalting column. The presence of eight free thiols was confirmed by the Ellman’s assay. To generate randomly oxidized disulfide bonds in scRNaseA, reduced RNaseA in 50 mM Tris–HCl pH 8.5 was incubated with 6 M Gdn-HCl in the dark at room temperature for 3 days before being acidified and purified. Oxidization of the disulfide bonds to produce scRNaseA was confirmed by the Ellman’s assay.
Isomerase activity of reduced Mt-DsbA and mutants were tested by measuring spectrophotometrically RNaseA cleavage of cyclic-2’,3’-cytidinemonophosphate (cCMP) to 3’-cytidinemonophosphate (3’CMP), which results in an increase in absorption at 296 nm. Purified Mt-DsbA or mutants (10 μM) were added to 100 mM sodium phosphate pH 7, 1 mM EDTA, and 10 μM DTT at room temperature for 5 minutes. To initiate the reaction, 40 μM scRNaseA was added. At several time points, 20 μl aliquots were taken and added to 60 μl of 4 mM cCMP, so that the final volume was 80 μl. The rate of RNaseA cleavage of cCMP was monitored at 296 nm for 3 min and the percentage of native RNaseA activity was plotted against time.
Disulfide bond forming proteins
Protein Data Bank
- vitamin K:
Gdn-HCl, guanidine hydrochloride
Matrix-assisted laser desorption/ionization time-of-flight
High-performance liquid chromatography
- scrambled RNaseA:
Single-wavelength anomalous diffraction
This work has been supported by the National Institutes of Health PO1-AI095208 (co-PI C.W.G). We thank the Advanced Light Source (ALS) at Berkeley National Laboratories and the Stanford Synchrotron Radiation Lightsource (SSRL) for their invaluable help in data collection. We would also like to thank Angelina Iniguez, Yama Latif and Seth T. Kazmer for their assistance in the project.
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