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Originally published In Press as doi:10.1074/jbc.M704044200 on October 3, 2007

J. Biol. Chem., Vol. 282, Issue 48, 34945-34951, November 30, 2007
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Crystal Structure of an Unusual Thioredoxin Protein with a Zinc Finger Domain*

Jiqing Ye{ddagger}§, Seung-Hyun Cho, Jessica Fuselier{ddagger}§, Weikai Li{ddagger}§, Jon Beckwith1, and Tom A. Rapoport, Howard Hughes Medical Investigator{ddagger}§2

From the {ddagger}Howard Hughes Medical Institute and the Departments of §Cell Biology and Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, May 16, 2007 , and in revised form, September 13, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Many Gram-negative bacteria have two cytoplasmic thioredoxins, thioredoxin-1 and -2, encoded by the trxA and trxC genes, respectively. Both thioredoxins have the highly conserved WCGPC motif and function as disulfide-bond reductases. However, thioredoxin-2 has unique features: it has an N-terminal motif that binds a zinc ion, and its transcription is under the control of OxyR, which allows it to be up-regulated under oxidative stress. Here, we report the crystal structure of thioredoxin-2 from Rhodobacter capsulatus. The C-terminal region of thioredoxin-2 forms a canonical thioredoxin fold with a central β-sheet consisting of five strands and four flanking {alpha}-helices on either side. The N-terminal zinc finger is composed of four short β-strands (S1–S4) connected by three short loops (L1–L3). The four cysteines are at loops L1 and L3 and form a tetragonal binding site for a zinc ion. The zinc finger is close to the first β-strand and first {alpha}-helix of the thioredoxin fold. Nevertheless, the zinc finger may not directly affect the oxidoreductase activity of thioredoxin-2 because the zinc finger is not near the active site of a protomer and because thioredoxin-2 is a monomer in solution. On the basis of structural similarity to the zinc fingers in Npl4 and Vps36, we propose that the N-terminal zinc finger of thioredoxin-2 mediates protein-protein interactions, possibly with its substrates or chaperones.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Oxidative stress causes the damage of DNA, protein, and lipid molecules and also affects their biosynthesis. All cells possess systems that serve as a defense against oxidative stress. These include reducing systems provided by GSH, glutaredoxins, and thioredoxins (1, 2). The thioredoxins are small cytoplasmic proteins present in all cells. They serve as reductases that reduce disulfide bonds in a wide range of proteins, including 3'-phosphoadenylylsulfate reductase, ribonucleotide reductase, methionine sulfoxide reductase, and the membrane protein DsbD. Thioredoxins have a highly conserved WCGPC motif; during the catalytic cycle, the two cysteines alternate between reduced and oxidized forms. When thioredoxin reduces a disulfide bridge, it itself becomes oxidized. The disulfide in thioredoxin must therefore be regenerated by a thioredoxin reductase, which in turn uses the reducing equivalents derived from NADPH (2, 3). In addition to functioning as an electron donor, thioredoxin is an essential component of the T7 DNA polymerase and is involved in the assembly of a number of filamentous viruses (4).

Bacterial thioredoxin-2 is a recently described cytoplasmic thioredoxin homolog. It shares ~30% overall sequence identity with thioredoxin-1 and contains an N-terminal extension of about ~40 residues (5–8). Thioredoxin-2 seems to play a role in the protection against oxidative stress, as the deletion of its gene in Rhodobacter capsulatus results in increased sensitivity to oxidative agents (8, 9). Furthermore, under oxidative stress conditions, the expression of thioredoxin-2 is up-regulated by the transcription factor OxyR (6, 9), whereas the expression of thioredoxin-1 does not change. The N-terminal extension contains two additional CXXC motifs and binds a zinc ion with high affinity. Based on in vitro experiments with the oxidative agent hydrogen peroxide, it has been proposed that the zinc ion is released from thioredoxin-2 under oxidative stress (7). However, it is not clear whether the zinc ion is released in intact cells particularly because thioredoxin-2 remains in its reduced state even under oxidative stress (6). Mutation of the zinc-binding domain does not affect the oxidoreductase activity of thioredoxin-2 in vivo, at least not for the substrates analyzed (6). However, it has been reported that the deletion of the zinc finger domain decreases the reductase activity in vitro (5). Thioredoxin-2 has been found in ~20 different bacterial species. Proteins with the same name in eukaryotes lack a zinc finger domain. Human thioredoxin-2, an ortholog of bacterial thioredoxin-1, resides in mitochondria. Yeast thioredoxin-2 is an almost identical isoform of cytosolic thioredoxin-1 (10, 11).

As the zinc finger of bacterial thioredoxin-2 is found in different organisms, it is unlikely to be without function. Similar domains are found in many proteins that are involved in DNA, RNA, or lipid binding; in protein-protein interactions; in protein stability; or in the sensing of oxidative stress (12). The zinc finger motif usually contains ~20–60 residues with cysteines and/or histidines folded into a tetragonal binding site for a zinc ion. The zinc ion is important in stabilizing the structural fold and binds with high affinity. Although all zinc finger domains share the tetragonal zinc coordination, their structures are as diverse as their functions. It is therefore not easy to predict the structure of a zinc finger domain on the basis of the amino acid sequence. Specifically, a BLAST search using thioredoxin-2 does not reveal any zinc fingers with known structures. The thioredoxin-2 zinc finger contains two CXXC motifs separated by 15–16 residues. A similar pattern is found in DksA (CXXCX17CXXC), transcription factor IIB (CXXCX15CXXC), and ClpX (CXXCX18CXXC) (13–16), but the structures of these zinc fingers are quite different. The DksA zinc finger has the typical ββ{alpha}-fold found in many transcription factors that bind directly to DNA; the transcription factor IIB zinc finger has four short strands that are involved in the interaction with RNA polymerase II; and the zinc finger in ClpX has two β-hairpins followed by an {alpha}-helix and is involved in homodimerization and substrate interaction (14, 15). It has been proposed that the zinc finger of thioredoxin-2 is similar to that in the redox sensor protein Hsp33 (7), but there is essentially no sequence similarity. The primary sequence of thioredoxin-2 does not reveal much of its function or structure.

Here, we report the crystal structure of R. capsulatus thioredoxin-2 at 1.92-Å resolution. Thioredoxin-2 has an N-terminal zinc finger and a C-terminal canonical thioredoxin fold. Our results show that the structure of the N-terminal zinc finger is similar to those of Npl4, Vps36, and transcription factor IIB (16–18). We propose that the zinc finger of thioredoxin-2 mediates protein-protein interactions with its substrates or chaperones during the defense against oxidative stress.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cloning and Protein Expression—The R. capsulatus thioredoxin-2 gene was amplified from genomic DNA by PCR and cloned into pET28a+ between the NdeI and HindIII sites. The recombinant protein has an N-terminal histidine tag and a thrombin site between the tag and thioredoxin-2. The expression vector was transformed into C43(DE3) cells. The transformed cells were grown to A600 = 0.7 and induced by the addition of isopropyl β-D-thiogalactopyranoside to a final concentration of 0.5 mM. Cells were harvested after culturing at 37 °C for an additional 4 h. The cell pellet was resuspended in Tris-buffered saline (20 mM Tris, 0.3 M NaCl, and 10% glycerol, pH 8.0), and cells were broken with a Microfluidizer. The recombinant thioredoxin-2 protein was in the supernatant and purified by Ni-NTA3 chromatography under native conditions. Thrombin was added to the purified sample at a ratio of 10 units of thrombin to 1 mg of recombinant thioredoxin-2. The cleaved sample was concentrated and further purified by size-exclusion fast protein liquid chromatography in a running buffer of 20 mM HEPES and 0.1 M NaCl, pH 7.4. Fractions containing thioredoxin-2 were pooled and concentrated to a final concentration ~10 mg/ml.

Crystallization, Data Collection, and Structure Determination—Before trays were set up, dithiothreitol was added to a final concentration of 5 mM, and the protein solution was incubated at room temperature for 1 h. Crystals were grown in 28–36% polyethylene glycol 400, 10 mM ZnSO4, and 50 mM N-(2-acetamido)iminodiacetic acid, pH 6.5, by the hanging drop method at 22 °C. Crystals usually grew to 200 x 200 x 50 µm3 in 1–2 weeks. Crystals were looped directly from the drop and flash-frozen in liquid nitrogen. Data were collected using synchrotron radiation at a zinc peak wavelength of 1.2826 Å at beam line ID19 of the Advanced Photon Source. A data set for single-wavelength anomalous diffraction (SAD) phasing was collected. The images were indexed and scaled by HKL3000 (19). Phasing, map calculation and improvement, and automated model building were done in HKL3000. Briefly, using the zinc SAD data set, the SHELX module in HKL3000 found three zinc sites (20). The zinc sites were refined and phases were calculated using mlphare (21). Automated model building was carried out using the ARP/wARP program (22). The electron density map was further improved by NCS averaging using the NCS operator derived from the partially built model (21). The map was used for a second round of ARP/wARP automated model building. Most of the model (~90%) was built. The rest was manually built using Coot (23). The refinement was carried out in CNS (24) against a second data set at 1.92-Å resolution.

Analytical Ultracentrifugation—Analytical ultracentrifugation was carried out on reduced thioredoxin-2 in a Beckman Coulter Optima XL-A ultracentrifuge. Three samples with initial concentrations of ~15, ~40, and ~80 µM thioredoxin-2 were loaded on a 6-well cell and centrifuged at three speeds of 24,000, 28,000, and 36,000 rpm. Data were collected after equilibrium was achieved as indicated by no changes in two successive scans separated by 4 h. Data were analyzed by Beckman XL-A data analysis software using MULTIFIT to an ideal self-association model (25).

Ni-NTA Pulldown of His-tagged Thioredoxin-2 and Identification of Its Binding Partner DnaK—Escherichia coli strain DHB4 ({Delta}(ara-leu)7697 araD139 {Delta}lacX74 galE galK rpsL phoR {Delta}(phoA)PvuII {Delta}malF3 thi) (26) was transformed with plasmid pDR1005 (pBAD33-His6-trxC) or pDR1006 (pBAD33-His6-{Delta}2–29trxC) or with the pBAD33 vector (6). Protein expression was induced by 0.2% L-arabinose at A600 = 0.1. Cells were grown at 37 or 42 °C after induction and harvested when the cell density reached A600 = 0.5. Cells from 10 ml of culture were lysed in 1 ml of buffer containing 20 mM Tris and 0.3 M NaCl, pH 8. The supernatant was mixed with 100 µl of Ni-NTA beads. The Ni-NTA beads were washed three times with 1 ml of the same buffer and then washed three times with 1 ml of the same buffer containing 30 mM imidazole. The proteins were eluted with 300 mM imidazole. Samples eluted from the Ni-NTA columns were run on SDS-polyacrylamide gel under reducing conditions. The bands in the SDS gel were cut out and analyzed by liquid chromatography/tandem mass spectrometry (Taplin Mass Spectrometry Facility, Harvard Medical School).


Figure 1
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  		FIGURE 1.
A, stereo view of the ribbon model of thioredoxin-2 (Protein Data Bank code 2PPT). The N-terminal zinc finger is shown in pink, and the thioredoxin fold in cyan. The zinc ion (shown in silver) is coordinated by four cysteines: Cys14, Cys17, Cys34, and Cys37. The side chains of cysteines (Cys73 and Cys76) at the active site are also highlighted. B, alignment of R. capsulatus thioredoxin-2 and E. coli thioredoxin. The secondary structure elements are indicated. C, overlay of thioredoxin-2 and thioredoxin structures (Protein Data Bank code 2TRX). The thioredoxin structure is shown in yellow. D, detailed view of the active sites of thioredoxin-2 (reduced form; left) and thioredoxin (oxidized form; right). The images were prepared using the program RIBBONS (39).

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Structure of Thioredoxin-2—R. capsulatus thioredoxin-2 was crystallized, and its structure was determined using SAD originating from the bound zinc ion (Table 1). Thioredoxin-2 has an N-terminal zinc finger motif and a C-terminal canonical thioredoxin fold (27). The thioredoxin fold consists of a five-stranded β-sheet with flanking {alpha}-helices (Fig. 1A). The secondary structural elements are β{alpha}β{alpha}β{alpha}ββ{alpha}, as shown in Fig. 1B. The distance between the sulfur atoms of Cys73 and Cys76 at the active site is 3.7 Å, indicating that, in the crystal, thioredoxin-2 is in its reduced state. This is expected because 5 mM dithiothreitol was added to the protein solution during crystallization. Nevertheless, the structure can be superimposed on the oxidized form of E. coli thioredoxin-1 with a backbone root mean square deviation of 0.93 Å (Fig. 1C), in agreement with the previous observation that the reduced and oxidized forms of thioredoxin-1 are almost identical (28). The active sites of thioredoxin-1 and -2 are also very similar (Fig. 1D). The highly conserved WCGPC motif is located at the loop following the central β-strand (β2) and the beginning of the second {alpha}-helix. The active-site cysteine (Cys73 in thioredoxin-2 and Cys32 in thioredoxin-1) is at the beginning of helix {alpha}2 and is solvent-exposed. This cysteine has a relatively low pKa (29). The deprotonated thiolate is presumably stabilized by the helical dipole moment of helix {alpha}2 (30). The second conserved cysteine (Cys76 in thioredoxin-2 and Cys35 in thioredoxin-1) has a higher pKa. It is likely to be deprotonated through a general base catalysis by an aspartic residue in the β2-strand (Asp67 in thioredoxin-2 and Asp26 in thioredoxin-1). Because the carboxyl group of this aspartic residue is ~5 Å away from the second sulfur atom, the deprotonation is probably mediated by the polarization of a water molecule. Another common feature among the thioredoxins is a positively charged residue (Arg77 in thioredoxin-2 and Lys36 in thioredoxin-1) that interacts with the backbone carbonyl group of the residue preceding the conserved tryptophan (Trp72 and Trp32, respectively). This interaction might be important to maintain the active-site conformation. Overall, thioredoxin-1 and -2 likely use the same mechanism to catalyze disulfide reduction of their substrates.


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  		TABLE 1
Data collection and refinement statistic

 
The N-terminal Zinc Finger—The N-terminal extension of thioredoxin-2 is folded into a zinc-binding finger, consisting of four short strands (S1–S4) connected by three short loops (L1–L3) (Fig. 2A). The S1/S2 strands are hydrogen-bonded with each other and connected by loop L1. The S3/S4 strands are shorter than the S1/S2 strands with fewer hydrogen bonds formed between them; they are connected by the short loop L3. The two CXXC motifs are located in the loops (L1 and L3) at the end of the four strands. The four cysteines form a tetragonal zinc-binding site. The zinc ion is clearly visible in the anomalous difference map (Fig. 2B). The S1/S2 strands are associated with the S3/S4 strands through the zinc ion and through hydrophobic interactions. A number of hydrophobic residues (Leu12 and Val23 in the S1/S2 strands and Pro32, Ile42, and Leu43 in the S3/S4 strands) are involved in packing these two structural elements together (Fig. 2A). These residues are relatively well conserved. Although the hydrophobic interactions are likely to be important, the zinc ion probably plays a dominant role in keeping the S1/S2 and S3/S4 structural elements together. The distance between the zinc ion and the sulfur atoms in the cysteines is <2.3 Å, indicating a strong interaction.


Figure 2
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  		FIGURE 2.
A, structure of the N-terminal zinc finger. Four cysteines (Cys14, Cys17, Cys34, and Cys37) that coordinate a zinc ion and residues involved in packing are shown in green. B, anomalous difference map contoured at 10{sigma}. Two zinc ions are shown in this protomer: one coordinated by the zinc finger and the second by Glu58 and His111.

 
In addition to the zinc ion in the zinc finger domain, a second zinc ion, coordinated by His111 and Asp58, is observed in one protomer in the crystal (Fig. 2B). The zinc ion at this site has a relatively low occupancy and is probably bound with low affinity.

The Zinc Finger Is Not Near the Active Site of Thioredoxin-2—In our structure, the zinc finger is not close to the WCGPC active site. Rather, it is located at one side of the thioredoxin fold defined by the first strand (β1) and the first {alpha}-helix ({alpha}1) (Fig. 1, A and B). The zinc ion is almost at the opposite end of the active site, with a distance of ~30 Å between them. It is thus quite unlikely that the zinc finger can directly affect the reactivity of the active site within the same molecule. It is also unlikely that the zinc finger would directly compete for substrate binding within a protomer. The known structures of complexes between a thioredoxin fold and a substrate (DsbD{alpha}·DsbD{gamma} (31), DsbA·DsbB (32), and thioredoxin·NF-{kappa}B (33)) indicate that the substrate-binding region is defined by the WCXXC active site at the β2-{alpha}2 region and the beginning of the β4-strand where the Gly-Ile-Pro conserved motif is located (Fig. 1D). These regions are far away from the zinc finger domain.


Figure 3
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  		FIGURE 3.
A, dimer of thioredoxin-2 in the asymmetric unit. The zinc finger and thioredoxin fold are shown in pink and cyan, respectively. Residues in the zinc finger (Leu15, Ala16, Cys17, Cys37, Gly38, and Ala39) involved in interaction with the thioredoxin fold are highlighted. The side chains of Gln102, Arg77, and the active-site residues Cys73 and Cys76 are shown in green. B, ultracentrifugation analysis. The fitting curves of two data sets of nine are shown: 40 µM thioredoxin-2 equilibrated at 28,000 rpm (lower left panel) and 36,000 rpm (lower right panel). The residuals of the fittings are plotted in the upper panels.

 
Dimer in Crystal and Monomer in Solution—Although the zinc finger is not close to the WCGPC active site or the substrate-binding groove within a protomer, it is conceivable that it could be close to one of these in an oligomer. In our crystals, the two protomers in the asymmetric unit are arranged such that the zinc finger of one protomer is close to the active site of the second protomer (Fig. 3A). Notably, the side chain of the conserved residue Arg77 forms a hydrogen bond with the backbone carbonyl of Cys37 in loop L3 of the zinc finger. In addition, the side chain of Gln102 is hydrogen-bonded with the carbonyl of Ala16 in loop L1. Finally, residues in loops L1 (Leu15, Ala16, and Cys17) and L3 (Cys37, Gly38, and Ala39) are in van der Waals contact with residues around the active site (Pro71, Trp72, Cys73, Pro75, and Arg77) (Fig. 3A). These interactions suggest that the zinc finger might affect the oxidoreductase activity of the thioredoxin fold in a dimer or higher oligomer. However, several lines of evidence suggest that thioredoxin-2 is monomeric in solution. First, the buried surface between the protomer is relatively small, ~1016 Å2 (7.5% of the total surface). Second, the reduced form of R. capsulatus thioredoxin-2 runs at the position of a monomer in gel filtration experiments (data not shown). Similar results have also been reported for both the reduced and oxidized forms of E. coli thioredoxin-2 (7). To exclude that thioredoxin-2 dimerizes with low affinity, we performed analytical ultracentrifugation experiments, which can measure Kd values as low as 1 mM. Thioredoxin-2 at three different concentrations (~15, ~40, and ~80 µM) was used and centrifuged to equilibrium at three different speeds. The data were fit to a self-association model. The best fit was obtained with a monomer species of 14.3 ± 0.6 kDa (theoretical molecular mass of 15.5 kDa) (Fig. 3B). The deviations from the theoretical values (Fig. 3B, upper panels) were close to zero and randomly distributed. The results indicate that thioredoxin-2 is a monomer in solution even at concentrations as high as 80 µM. The dimer observed in the crystal is thus likely to be a non-physiological artifact. It should be noted that in other thioredoxin crystals, dimers or oligomers were also observed in the asymmetric unit (34–36). Given that the interfaces between the monomers are different in these structures and that the existence of dimers or oligomers in solution has not been demonstrated for any of the wild-type thioredoxins, it appears that all members of the family function as monomers.


Figure 4
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  		FIGURE 4.
A, overlay of the zinc fingers of thioredoxin-2, Vps36, and Npl4. The zinc fingers of Vps36 (blue) and Npl4 (yellow) are superimposed onto that of thioredoxin-2 (pink). The zinc ions are also colored the same way as their respective ribbon model. The side chains of thioredoxin-2 cysteines are shown in green. B, binding determinants in thioredoxin-2 (left), Vps36 (middle), and Npl4 (right). The zinc fingers are oriented as described for A. The side chains of residues involved in protein-protein interactions and the cysteines in zinc ion coordination are shown in green. The zinc ions are shown in silver. C, the second protein-protein interaction region of the thioredoxin-2 zinc finger in the crystal. The extended N terminus of one protomer (shown in green) binds to the side of the zinc finger of a symmetry-related protomer (shown in pink). Met2 and Lys5 of the N terminus form hydrogen bonds (shown as blue dashed lines) with Ala20, Lys22, and Lys33 (shown in gray).

 
The Zinc Finger of Thioredoxin-2 Is Homologous to Those That Mediate Protein-Protein Interactions—We performed a Dali search using the zinc finger of thioredoxin-2 (residues 7–44) to find homologous structures. Four structures (Vps36 (Protein Data Bank code 2J9U), Npl4 (code 1NJ3), 30 S ribosomal protein S27e (code 1NVH), and YAF2 (code 2D9G)) were found with Z scores >2 (37). The structures are considered to be similar if the Dali Z score is >2. All of them contain two CXXC motifs that form a tetragonal zinc-binding site. The length between the CXXC motifs varies from 10 residues in Npl4 to 16 residues in thioredoxin-2. To illustrate the structural similarity, the zinc fingers of Vps36 (code 2J9U) and Npl4 (code 1NJ3) were superimposed on that of thioredoxin-2 with an overall backbone root mean square deviation of ~1.9 Å (Fig. 4A) (16–18). As shown in Fig. 4A, the positions of the zinc ions are very close to each other. Loop L2 is short in Npl4 (shown in yellow) and relatively long in thioredoxin-2 and Vps36 (shown in pink and blue, respectively), yet the overall structure at the zinc-binding site is similar.

Among the four proteins, the functions of two zinc fingers (Vps36 and Npl4) have been well studied. Vps36 is a component of the ESCRT-II complex (endosomal sorting complex required for transport) that is involved in the sorting and transport of ubiquitinated proteins to multivesicular bodies. The Vps36 zinc finger binds to Vps28, an ESCRT-I component, with high affinity (nanomolar range). Residues involved in the association (Ile122, Cys123, Asn145, and Cys146) are located in two loops (L1 and L3) of the zinc finger domain (Fig. 4B) (17). Mutations in the binding interface abolish the binding to Vps28. The zinc finger of Npl4 has been shown to bind to ubiquitin with low affinity (with a Kd of ~100–200 µM). A conserved TF (Thr13-Phe14) motif immediately following the first CXXC motif has been implicated in ubiquitin binding as shown by chemical shift perturbation and mutagenesis studies. In addition, Met25 near the second CXXC motif is also important for ubiquitin binding. The TF binding surface is not exactly the same as that of Vps36 (Fig. 4B) (16). Nevertheless, both are located at loops L1 and L3.

We noticed two surfaces of the thioredoxin-2 zinc finger that are involved in protein-protein contacts in the crystal. The first binding surface is the one in the dimerization interface (Figs. 3A and 4B). This binding surface resembles that of Vps36 and, to a lesser extent, that of Npl4. The second binding surface is shown in Fig. 4C. It involves the unstructured N terminus (residue 1–7) of one protomer interacting with one side of the zinc finger domain of a symmetry-related molecule. Met2 and Lys5 in the N-terminal segment form four hydrogen bonds with Ala20, Lys22, and Lys33 in the zinc finger (Fig. 4C). On the basis of these interactions and the similarity to other zinc finger domains, we propose that this type of zinc finger mediates protein-protein interactions. Specifically, the zinc finger of thioredoxin-2 may be involved in the binding of substrates and/or chaperones. Thioredoxin-2 has been found to interact with a number of proteins, including the DnaJ and DnaK chaperones, in a large-scale search for interacting proteins in E. coli (38). Our attempts to confirm the interaction with DnaJ by direct pulldown experiments have been unsuccessful (data not shown). However, the interaction with DnaK could be confirmed by us in experiments in which we pulled down His-tagged thioredoxin-2 with Ni-NTA beads and identified bound proteins by mass spectrometry (Fig. 5). A thioredoxin-2 mutant lacking the zinc domain appeared to bind less DnaK (Fig. 5, lane 3 versus lane 4). These data suggest that the zinc domain may interact with the DnaK chaperone, but they do not exclude the possibility that a fraction of this domain may be unfolded and thus be bound by DnaK.


Figure 5
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  		FIGURE 5.
Interaction of thioredoxin-2 with DnaK. His-tagged wild-type thioredoxin-2 (pDR1005; lanes 2 and 3) and a mutant with the zinc finger domain deleted (pDR1006; lane 4) were purified from cells grown at 37 or 42 °C using Ni-NTA. A mock purification was also included (lane 1) using cells transformed with the pBAD33 vector. Proteins were visualized by Coomassie Blue staining. A band at ~70 kDa (indicated by the asterisk) in lane 3 and the corresponding region in lane 4 were cut out and analyzed by mass spectrometry. For lane 3, the recovered peptides are predominantly derived from DnaK (41 peptides). In comparison, only nine peptides of DnaK were recovered from lane 4. Peptides derived from nonspecific bound proteins such as glucosamine synthetase (GlmS; 32 versus 30 peptides) and the heat shock protein HtpG (27 versus 32 peptides) are comparable for lanes 3 and 4.

 
The zinc finger of thioredoxin-2 is different from that of Hsp33, a redox sensor, and from those of DNA-binding proteins. These results indicate that thioredoxin-2 does not act as a redox sensor, is not involved in protecting DNA against oxidative damage, and is not involved in transcription, yet conservation of this domain suggests that it has an important function. On the basis of the structural homology to the zinc fingers of Npl4 and Vps36, we propose that the zinc finger of thioredoxin-2 mediates protein-protein interactions.

Thioredoxin-2 Is Conserved among Proteobacteria, and Its Zinc Finger Is Seen in Other Proteins—Although thioredoxin-2 was initially found only in E. coli and Corynebacterium nephridii (5), we found 85 homologs using the zinc finger domain (residues 1–40) of the protein from R. capsulatus in a BLAST search. All of these proteins are from Gram-negative proteobacteria, representing all five ({alpha}–{epsilon}) classes. The only exception is the protein from C. nephridii, a Gram-positive actinobacterium. We then performed a pattern search using CXXCX10–20CXXCX30–40WCXXC in which we allowed 10–20 residues between the two CXXC motifs in the zinc finger and 30–40 residues between the second CXXC motif and the WCXXC active site of thioredoxin. We identified 12 additional homologs from Gram-negative proteobacteria, four from Gram-positive actinobacteria (Streptomyces avermitilis, Arthrobacter aurescens, Mycobacterium vanbaalenii, and Mycobacterium flavescens), and one from a Deinococcus-Thermus species (Thermus thermophilus). Thus, thioredoxin-2 is conserved in >100 different bacteria. It is interesting that a zinc finger motif homologous to that of thioredoxin-2 is also present in the Paraquat-inducible membrane protein Pqi and in a diguanylate cyclase/phosphodiesterase from Marinobacter aquaeolei VT8. The zinc finger may thus have evolved from a common ancestor and be used in various proteins as a functional module. This is consistent with our proposal that the N-terminal zinc finger is involved in protein-protein interactions.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2PPT) 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 in part by National Institutes of Health Grants GMO41883 (to J. B.) and GM052586 (to T. A. R.). 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

1 American Cancer Society Professor. Back

2 To whom correspondence should be addressed: Dept. of Cell Biology, Howard Hughes Medical Inst., Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1612; Fax: 617-432-1190; E-mail: tom_rapoport{at}hms.harvard.edu.

3 The abbreviations used are: Ni-NTA, nickel-nitrilotriacetic acid; SAD, single-wavelength anomalous diffraction. Back


   ACKNOWLEDGMENTS
 
We thank P. Sliz for organizing synchrotron trips. We are grateful to our colleagues at beam line ID19 of the Advanced Photon Source for invaluable help. We thank Drs. J. Al-Bassam and S. Harrison for help in the analytical ultracentrifugation experiment. We thank Dr. B. Burton for discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Oktyabrsky, O. N., and Smirnova, G. V. (2007) Biochemistry (Mosc.) 72, 132-145[CrossRef][Medline] [Order article via Infotrieve]
  2. Ritz, D., and Beckwith, J. (2001) Annu. Rev. Microbiol. 55, 21-48[CrossRef][Medline] [Order article via Infotrieve]
  3. Arner, E. S., and Holmgren, A. (2000) Eur. J. Biochem. 267, 6102-6109[Medline] [Order article via Infotrieve]
  4. Russel, M., and Model, P. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 29-33[Abstract/Free Full Text]
  5. Miranda-Vizuete, A., Damdimopoulos, A. E., Gustafsson, J., and Spyrou, G. (1997) J. Biol. Chem. 272, 30841-30847[Abstract/Free Full Text]
  6. Ritz, D., Patel, H., Doan, B., Zheng, M., Aslund, F., Storz, G., and Beckwith, J. (2000) J. Biol. Chem. 275, 2505-2512[Abstract/Free Full Text]
  7. Collet, J. F., D'Souza, J. C., Jakob, U., and Bardwell, J. C. (2003) J. Biol. Chem. 278, 45325-45332[Abstract/Free Full Text]
  8. Li, K., Hartig, E., and Klug, G. (2003) Microbiology (Read.) 149, 419-430[Abstract/Free Full Text]
  9. Zeller, T., Li, K., and Klug, G. (2006) J. Bacteriol. 188, 7689-7695[Abstract/Free Full Text]
  10. Gan, Z. R. (1991) J. Biol. Chem. 266, 1692-1696[Abstract/Free Full Text]
  11. Smeets, A., Evrard, C., Landtmeters, M., Marchand, C., Knoops, B., and Declercq, J. P. (2005) Protein Sci. 14, 2610-2621[Abstract/Free Full Text]
  12. Laity, J. H., Lee, B. M., and Wright, P. E. (2001) Curr. Opin. Struct. Biol. 11, 39-46[CrossRef][Medline] [Order article via Infotrieve]
  13. Perederina, A., Svetlov, V., Vassylyeva, M. N., Tahirov, T. H., Yokoyama, S., Artsimovitch, I., and Vassylyev, D. G. (2004) Cell 118, 297-309[CrossRef][Medline] [Order article via Infotrieve]
  14. Donaldson, L. W., Wojtyra, U., and Houry, W. A. (2003) J. Biol. Chem. 278, 48991-48996[Abstract/Free Full Text]
  15. Zhu, W., Zeng, Q., Colangelo, C. M., Lewis, M., Summers, M. F., and Scott, R. A. (1996) Nat. Struct. Biol. 3, 122-124[CrossRef][Medline] [Order article via Infotrieve]
  16. Wang, B., Alam, S. L., Meyer, H. H., Payne, M., Stemmler, T. L., Davis, D. R., and Sundquist, W. I. (2003) J. Biol. Chem. 278, 20225-20234[Abstract/Free Full Text]
  17. Teo, H., Gill, D. J., Sun, J., Perisic, O., Veprintsev, D. B., Vallis, Y., Emr, S. D., and Williams, R. L. (2006) Cell 125, 99-111[CrossRef][Medline] [Order article via Infotrieve]
  18. Teo, H., Perisic, O., Gonzalez, B., and Williams, R. L. (2004) Dev. Cell 7, 559-569[CrossRef][Medline] [Order article via Infotrieve]
  19. Minor, W., Cymborowski, M., Otwinowski, Z., and Chruszcz, M. (2006) Acta Crystallogr. Sect. D Biol. Crystallogr. 62, 859-866[CrossRef][Medline] [Order article via Infotrieve]
  20. Uson, I., and Sheldrick, G. M. (1999) Curr. Opin. Struct. Biol. 9, 643-648[CrossRef][Medline] [Order article via Infotrieve]
  21. Collaborative Computational Project 4 (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  22. Perrakis, A., Morris, R., and Lamzin, V. S. (1999) Nat. Struct. Biol. 6, 458-463[CrossRef][Medline] [Order article via Infotrieve]
  23. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. Sect. D. Biol. Crystallogr. 60, 2126-2132[CrossRef][Medline] [Order article via Infotrieve]
  24. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D. Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
  25. Lebowitz, J., Lewis, M. S., and Schuck, P. (2002) Protein Sci. 11, 2067-2079[Abstract/Free Full Text]
  26. Boyd, D., Manoil, C., and Beckwith, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8525-8529[Abstract/Free Full Text]
  27. Katti, S. K., LeMaster, D. M., and Eklund, H. (1990) J. Mol. Biol. 212, 167-184[CrossRef][Medline] [Order article via Infotrieve]
  28. Jeng, M. F., Campbell, A. P., Begley, T., Holmgren, A., Case, D. A., Wright, P. E., and Dyson, H. J. (1994) Structure (Lond.) 2, 853-868[Medline] [Order article via Infotrieve]
  29. Takahashi, N., and Creighton, T. E. (1996) Biochemistry 35, 8342-8353[CrossRef][Medline] [Order article via Infotrieve]
  30. Holmgren, A. (1995) Structure (Lond.) 3, 239-243[Medline] [Order article via Infotrieve]
  31. Rozhkova, A., Stirnimann, C. U., Frei, P., Grauschopf, U., Brunisholz, R., Grutter, M. G., Capitani, G., and Glockshuber, R. (2004) EMBO J. 23, 1709-1719[CrossRef][Medline] [Order article via Infotrieve]
  32. Inaba, K., Murakami, S., Suzuki, M., Nakagawa, A., Yamashita, E., Okada, K., and Ito, K. (2006) Cell 127, 789-801[CrossRef][Medline] [Order article via Infotrieve]
  33. Qin, J., Clore, G. M., Kennedy, W. M., Huth, J. R., and Gronenborn, A. M. (1995) Structure (Lond.) 3, 289-297[Medline] [Order article via Infotrieve]
  34. Bao, R., Chen, Y., Tang, Y. J., Janin, J., and Zhou, C. Z. (2007) Proteins 66, 246-249[CrossRef][Medline] [Order article via Infotrieve]
  35. Amorim, G. C., Pinheiro, A. S., Netto, L. E., Valente, A. P., and Almeida, F. C. (2007) J. Biomol. NMR 38, 99-104[CrossRef][Medline] [Order article via Infotrieve]
  36. Capitani, G., Markovic-Housley, Z., DelVal, G., Morris, M., Jansonius, J. N., and Schurmann, P. (2000) J. Mol. Biol. 302, 135-154[CrossRef][Medline] [Order article via Infotrieve]
  37. Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123-138[CrossRef][Medline] [Order article via Infotrieve]
  38. Butland, G., Peregrin-Alvarez, J. M., Li, J., Yang, W., Yang, X., Canadien, V., Starostine, A., Richards, D., Beattie, B., Krogan, N., Davey, M., Parkinson, J., Greenblatt, J., and Emili, A. (2005) Nature 433, 531-537[CrossRef][Medline] [Order article via Infotrieve]
  39. Carson, M. (1997) Methods Enzymol. 277, 493-505[Medline] [Order article via Infotrieve]

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