Structure of Mycobacterium tuberculosis thioredoxin C

Mycobacterium tuberculosis is a facultative intracellular parasite of alveolar macrophages. M. tuberculosis is able to propagate in harsh environments within cells such as phagocytes, despite being exposed to reactive oxygen and nitrogen intermediates. The thioredoxin redox system is conserved across the phyla and has a well characterized role in resisting oxidative stress and influencing gene expression within prokaryotic and eukaryotic cells. M. tuberculosis thioredoxin (MtbTrx) has similar functions in redox homeostasis and it has recently been shown that alkyl hydroperoxidase C is efficiently reduced to its active form by MtbTrxC, supporting this notion. To address whether the MtbTrx has similar features to other thioredoxin structures and to examine the opportunities for designing drugs against this target, MtbTrxC has been crystallized and its structure determined to 1.3 Å resolution. Unexpectedly, the structure demonstrates an interesting crystal packing in which five C-­terminal residues from the MtbTrxC fold insert into a groove adjacent to the active site. A very similar interaction is observed in structures of human thioredoxins bound to peptides from the target proteins NF-κB and Ref-1.

1. Introduction

Mycobacterium tuberculosis is one of the most devastating pathogens and is thought to annually infect an estimated eight million people, resulting in 2–3 million deaths (Dye et al., 1999). The pathogenesis of M. tuberculosis still remains an expanding global health problem that compels new thera­peutic and preventative measures, with the emergence of multi-drug-resistant strains creating a worldwide emergency (Glickman & Jacobs, 2001). The resistance of the organism towards the host immune response and its ability to survive and reactivate at a later stage is poorly understood (Akif et al., 2005). An obligate aerobe, M. tuberculosis resides within alveolar mononuclear phagocytes and is thought to use a variety of redox systems to protect itself against the oxidative intermediates generated by the macrophages (Shinnick et al., 1995). Fernando et al. (1992) have shown that the thioredoxin system can also aid in the regeneration of inactivated proteins caused by the oxidative stress.

The thioredoxin system, consisting of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, is a ubiquitous redox pathway found in a wide variety of phyla (Holmgren, 1985, 1989). TrxR catalyses the reduction of Trx, which when present in its dithiol form is the main disulfide reductase in cells (Williams et al., 2000). This redox system has a wide variety of biological functions, including maintaining an intracellular reduced state in the face of an oxidizing extracellular environment (Powis et al., 1995). Trx and TrxR within M. tuberculosis are likely to play a similar key role resisting oxidative stress as observed in other prokaryotes, thus making thioredoxin a target for anti-M. tuberculosis drugs (Zhang et al., 1999). The M. tuberculosis genome encodes three thioredoxin proteins (TrxA, TrxB and TrxC), with the first biochemical inhibitors of M. tuberculosis thioredoxin C (MtbTrxC) signalling recently being reported (Shah et al., 2006). Since mycobacteria are devoid of the glutathione-dependent detoxification system, the thioredoxin system should significantly contribute towards the pathogen's defence against oxidative intermediates (Cole et al., 1998) and it has been shown that MtbTrxC dominates this detoxification pathway (Jaeger et al., 2004). In addition, alkyl hydroperoxidase (ahpC), which is another system known to combat oxidative stress in M. tuberculosis, is shown to require MtbTrxC to reduce it before it becomes catalytically active (Manca et al., 1999).

Thioredoxin proteins are highly conserved across prokary­otes and eukaryotes, having sequence identities of between 27 and 69% (Eklund et al., 1991). MtbTrxC has 50 and 29% sequence identity to E. coli and human thioredoxins, respectively. We report the crystal structure of MtbTrxC determined to 1.3 Å resolution, showing a novel crystal packing with five C-terminal residues overlaying the hydrophobic groove of the redox-active site.

2. Materials and methods

2.1. Cloning, expression and purification

The open reading frame for M. tuberculosis TrxC (Rv3914) was amplified from genomic DNA, kindly supplied by T. H. M. Ottenhoff (Leiden University Medical Centre, Leiden, The Netherlands), using standard PCR protocols and the resultant product was cloned into the expression vector pTrcHisA (Invitrogen). This plasmid was transformed into Escherichia coli BL21 (DE3) cells for expression. Cultures were grown at 310 K until an OD₅₉₅ of 0.6 was reached, prior to being induced with isopropyl β-D-thiogalactopyranoside (IPTG) and left to incubate overnight at 295 K. The cells were harvested by centrifugation at 4500g and resuspended in 20 mM Tris–HCl pH 7.9, 5 mM imidazole and 0.5 M NaCl prior to lysis by sonication (Branson). His-tagged MtbTrxC was purified from the supernatant by Ni²⁺-affinity chromatography using a pre-charged nickel-chelate affinity resin (Novagen). The protein was eluted using 20 mM Tris–HCl pH 7.9, 1 M imidazole and 0.5 M NaCl, with the MtbTrxC fractions pooled together and dialysed overnight in 1 l 20 mM Tris–HCl pH 7.9 and 0.5 M NaCl to remove the imidazole. The His tag was cleaved using enterokinase (Sigma) at an enzyme:substrate ratio of 1:100(w:w), resulting in an extra five N-terminal residues Asp, Arg, Trp, Gly and Ser prior to the start of the native protein sequence. After digestion, the protein was concentrated to a volume of 5 ml and loaded onto a HiLoad Superdex 75 gel-filtration column (Amersham Biosciences) pre-equilibrated with 20 mM Tris–HCl pH 7.9 and 0.5 M NaCl. Fractions of the protein were collected and analysed by SDS–PAGE before being pooled and concentrated (Vivaspin column) to 3 mg ml⁻¹.

2.2. Crystallization and data collection

Crystals grew in 10% 2-propanol, 0.1 M Na HEPES pH 7.5 and 20% PEG 4K using sitting-drop vapour diffusion at 293 K with a total drop volume of 2.0 µl. The crystals belonged to space group P1, with unit-cell parameters a = 26.48, b = 29.23, c = 30.95 Å, α = 88.37, β = 88.15, γ = 66.67°. A single crystal was flash-frozen using liquid nitrogen in mother liquor plus 20% glycerol. An initial data set was collected to 2.0 Å using a Rigaku Micromax-007 X-ray generator (λ = 1.5418 Å) and R-­AXIS IV⁺⁺ image plate and a subsequent data set was collected to 1.3 Å resolution at the European Synchrotron Research Facility (ESRF) using beamline ID14-2 (λ = 0.9792 Å). As a result of the crystal form, a number of unique reflections are too close to the rotation axis and are subsequently lost. Therefore, both the high- and low-resolution data sets were indexed and integrated separately using MOSFLM (Leslie, 1999) before being merged and then scaled using SCALA (Evans, 1993) from the CCP4 program suite (Collaborative Computational Project, Number 4, 1994), giving an overall R_merge of 0.178 and a completeness of 94.7% (Table 1).

Table 1
Crystallographic statistics

Values in parentheses are for the highest resolution shell.

Space group	P1
Unit-cell parameters (Å, °)	a = 26.48, b = 29.23, c = 30.95, α = 88.37, β = 88.15, γ = 66.67
Resolution limits (Å)	18.90–1.30 (1.37–1.30)
No. of observed/unique reflections	22036/11739
Completeness (%)	2.1 (2.0)
Data redundancy	94.7 (92.3)
Rmerge (%)	17.8 (22.6)
Mean I/σ(I)	8.7 (4.2)
Rwork/Rfree (%)	21.2 (28.3)/24.0 (34.1)
R.m.s. bond lengths (Å)	0.009
R.m.s. bond angles (°)	1.3
No. of protein residues	108
No. of water molecules	159
Average B factor, protein (Å^2)	8.5
R.m.s. B, protein (Å^2)	0.7
Average B factor, solvent (Å^2)	20.9
Residues in Ramachandran plot regions (%)
Most favoured	95.8
Additional allowed	4.2

2.3. Structure determination and refinement

A homology model of MtbTrxC was produced from the E. coli thioredoxin crystal structure (Katti et al., 1990; PDB code 2trx) using SWISSMODEL (Peitsch & Jongeneel, 1993). The MtbTrxC structure was solved by molecular replacement with Phaser (McCoy et al., 2005) using the homology model, locating one molecule in the asymmetric unit. The highest peak in the cross-rotation function gave the correct orientation of the monomer and an R factor of 0.38. The resulting 2F_o − F_c map gave clear electron density, allowing model building of 108 residues. Subsequent refinement took place using REFMAC5 (Murshudov et al., 1997), with intermittent cycles of manual model correction using Coot (Emsley & Cowtan, 2004). A total of 159 water molecules and seven dual rotamers were modelled using Coot to give a final model that has an R factor of 0.212 and R_free = 0.240, with 95.8% of residues in the most favoured region and the remainder in the additional allowed area of the Ramachandran plot. Seven N-­terminal residues and two C-terminal residues have no interpretable electron density and are assumed to be disordered.

3. Results and discussion

3.1. Molecular structure of M. tuberculosis thioredoxin C

M. tuberculosis thioredoxin C has the characteristic thioredoxin fold consisting of a five-stranded β-­sheet, three strands of which are parallel and two antiparallel, that forms a hydrophobic core surrounded by four α-helices (Fig. 1). The active site of MtbTrxC is located in the loop region between helix α2 and strand β2 and has a conformation identical to those of other oxidized thioredoxins. The two active-site cysteine residues Cys37 and Cys40 are found at the end of the α2 helix, with the Cys40 residue buried more significantly compared with Cys37. The published crystal structure of human thioredoxin (Weichsel et al., 1996; PDB code 1ert) demonstrates dimer formation, which is primarily mediated by a disulfide bridge between Cys73 residues, but also includes numerous hydrophobic contacts and a hydrogen bond between symmetrically related Asp60 residues. Although the dimer has also been observed with a Cys73→Ser mutation, suggesting that the other noncovalent interactions are important, this crystal packing is not seen with other thioredoxin structures. The crystal structure of MtbTrxC does not show any similar dimeric arrangements as observed in the human structures, even though the hydrophobic residues in the region are highly conserved and the relevent aspartic acid residue is still present.

Figure 1 Ribbon diagram portraying the secondary structure and fold of MtbTrxC. The crystal packing present in the primitive triclinic unit cell of MtbTrxC is also shown, portraying the C-terminal tail packed into the active-site groove of a symmetry mate. Figures were prepared using PyMOL (DeLano, 2002).

3.2. C-terminal polypeptide tail

A remarkable feature of the MtbTrxC structure is that the five C-terminal residues, consisting of Asp110, Val111, Val112, Pro113 and Asn114, lie in the active-site groove of an adjacent molecule in the crystal (Fig. 1). These residues might have been expected to continue the α4 helix, with the three hydrophobic residues Val111, Val112 and Pro113 interacting with the end of the β4 strand and the start of the α5 helix, as seen in other thioredoxin crystal structures. The interaction is comprised of significant hydrophobic and hydrogen-bonding contacts. The hydrophobic packing interactions occur between Pro113 and Trp35 and between Val111 and Val65, Val77 and Ile80, with Val111 sitting in a hydrophobic pocket at the end of the groove. Three hydrogen bonds of less than 3.2 Å feature in the polypeptide interactions: between Ile80 amide and Val108 carbonyl oxygen, Ile80 carbonyl oxygen and Asn110 amide and Ser79 hydroxyl oxygen and Asn110 side-chain amide group. All of these contacts allow the C-terminal polypeptide tail to follow the curvature of the protein and rest between the loop regions of β2–α2 and α3–β4 in a hydrophobic groove that measures 3–4 Å in depth, 3–4 Å in breadth and 11–12 Å in length. Instances of residues from symmetry-related molecules packing into the active site of thioredoxin and thioredoxin-like structures have been reported before in Anabaena thioredoxin (Saarinen et al., 1995; PDB code 1thx) and in tryparedoxins (Alphey et al., 2003; PDB code 1o73). However, in both examples the interaction involves soft crystal contacts containing one or two residues, whereas the C-terminal tail found in MtbTrxC is seen to follow the curvature of the structure along the redox-active groove and involves more than five residues.

A polypeptide chain interacting with this hydrophobic groove has also been observed in NMR structures of human thioredoxin and residues from its ligand-binding partners NF-­κB and Ref-1 (Qin et al., 1995, 1996). Although both of these proteins use cysteine residues to covalently interact with the active site of the thioredoxin, they have local polypeptide sequences that somewhat resemble the MTbTrxC tail. Using a nomenclature similar to that described by Qin et al. (1995, 1996), where the residue of the binding polypeptide adjacent to active-site cysteine (Cys32 for hTrx, Cys37 for MtbTrxC) is designated S₀, it is possible to describe some important similarities that may give rise to the interaction seen in our structure. For the human thioredoxin-NF-κB complex, S₀ is cysteine, whereas for the polypeptide tail observed in MtbTrxC S₀ is proline (Fig. 2).

Figure 2 (a) Sequence alignment of the polypeptide tail and NF-κB residues interacting with thioredoxin. (b) Stereoviews portraying the alignment of the five C-terminal residues of the tail of MtbTrxC and (c) five residues of the thioredoxin binding partner NF-κB from the NMR structure 1mdi (Qin et al., 1995), resting in their respective thioredoxin active sites. The residues lining the hydrophobic pockets are also shown.

Qin et al. (1995, 1996) describe an important interaction made by the side chain of a hydrophobic residue at position S₋₂, which sits in a hydrophobic pocket on the surface of thioredoxin. In the NF-κB and Ref-1 complexes the residue is tyrosine and tryptophan, respectively, and the associated pocket (in human thioredoxin) is formed by residues Phe27, Val59, Ala66, Val71 and Met74. In comparison, the MtbTrxC crystal structure has Val111 in the S₋₂ position and the hydrophobic pocket is formed by residues Phe32, Val65, Ala72, Val77 and Ile80. Thus, we see that the amino acids from the respective thioredoxins involved in these hydrophobic interactions are highly conserved, although owing to its smaller size there are fewer residues found interacting with Val111 of the MtbTrxC polypeptide tail. In fact, most of the residues conserved between all thioredoxins, outside of the active site, are found around this hydrophobic pocket (Fig. 3). The most highly conserved feature across the three complexes is a hydrogen bond that exists between the backbone N atom of the S₁ residue of the binding peptide and the carbonyl O atom of either Thr74 (human) or Ile80 (MtbTrxC) (Fig. 2). This could represent a conserved feature of thioredoxin protein–protein interactions when functioning as a redox protein, signalling modulator or chaperone.

Figure 3 (a) Molecular surface and (b) ball-and-stick diagrams portraying the sequence conservation between human thioredoxin and M. tuberculosis thioredoxin C around the active-site groove, where red shows high conservation and yellow shows low conservation. (c) Sequence alignment of MtbTrxC and human thioredoxin (hTrx), with conserved residues coloured in the same scheme and the active site in bold.

4. Conclusions

The structure of MtbTrxC reveals a five-residue polypeptide tail packing into the shallow groove that contains the active-site cysteine residues of a neighbouring molecule. This interaction undoubtedly arises as a result of crystallization, but it is nevertheless structurally very similar to the protein–protein interactions formed by human thioredoxin with the ligands NF-κB and Ref-1 (Qin et al., 1995, 1996).

As thioredoxin is noted for its solubility, a predominantly hydrophobic outlying region, such as these five C-terminal residues, disrupts the tight and compact nature of the thioredoxin fold. The question raised is whether the extended polypeptide tail exists in the solution structure of MtbTrxC and whether it has a discrete function. It seems likely that the polypeptide-tail conformation seen here is a component of the crystallization process and in its natural state the C-­terminal residues would continue the fourth α-helix as predicted. If this is the case, then it must be assumed that the energy required to partially unwind the α-helix is compensated by the stable interactions formed between the polypeptide tail and the adjacent protein in the crystal during the crystallization process.

Although arguably artificial, the polypeptide-tail interaction, along with those found between human thioredoxin and its target proteins, allows us to gain a better understanding of the binding interactions that can occur within the active site. It may also be potentially used to allow us to understand the peptide sequence that thioredoxin recognizes within these target proteins. This will enable us to predict the target regions that thioredoxin recognizes in proteins known to interact with this redox protein.

This conserved interaction motif indicates the likelihood that prokaryotic thioredoxins will also utilize this groove to interact with ligands in a manner reminiscent of human thioredoxin. In E. coli, a proteome analysis resulted in the identification of a total of 80 proteins found to be associated with the E. coli Trx1, implicating thioredoxin in at least 26 distinct cellular processes, including cell division, transcriptional regulation, energy transduction, protein folding and degradation, and several biosynthetic pathways (Kumar et al., 2004; Zeller & Gabriele, 2006). The exact ligand-binding partners for MtbTrxC are currently unknown, but they are likely to be linked to redox homeostasis and gene regulation and may form conserved interactions utilizing the thioredoxin groove in a manner reminiscent of the crystal structure packing observed here.