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Introduction

  1. Top of page
  2. Introduction
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Thioredoxin (TRX) is a well-known small enzyme that is present in all living organisms. It was first described in 1964 by Laurent et al.1 as a redox protein with a conserved active-site sequence, Trp–Cys–Gly(Ala)–Pro–Cys, that catalyzes many redox reactions through the reversible oxidation of dithiol to a disulfide. TRX has long been employed as a model system for studying protein stability because of its globular shape, small size, and high thermostability.2–5 The open reading frame of ST2123 from thermophilic archaea Sulfolobus tokodaii strain7, consisting of 140 amino acid residues with a molecular mass of 15.7 kDa, was annotated as a hypothetical TRX protein. A PSI-BLAST search, using the amino acid sequence of ST2123 as the query, identified from 37 to 140 amino acid residues, has up to 40% of similarity to the thioreoxins of known structure. Here we report the first crystal structure of archaeal TRX of ST2123 from S. tokodaii at 1.49 Å resolution and discuss the characteristic features of this thermostable protein.

Materials and methods

  1. Top of page
  2. Introduction
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Protein expression and purification

The ST2123 gene was amplified by PCR using S. tokodaii strain7 genomic DNA as a template. K53E mutant was obtained unexpectedly by random mutation during the PCR process. Amplified fragments were digested with NdeI and BamHI, and cloned into the pET28a expression vector (Novagen). An Escherichia coli overexpression strain, Rosetta (DE3) (Novagen), was transformed by the expression vectors containing the K53E mutant of ST2123 genes, and was cultivated at 37°C. The protein expression was induced by 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG). The bacterial cells were harvested by centrifugation and resuspended in 50 mM phosphate buffer (pH 8.0), containing 300 mM NaCl and 10 mM imidazole, and disrupted by sonication. After centrifugation, the supernatant was purified by heat treatment at 80°C for 30 min and a Ni-NTA agarose column (QIAGEN). The purified solution was dialyzed against 20 mM Tris–HCl buffer (pH 8.0) and treated with thrombin (GE healthcare) for digestion of the N-terminal His-tag. The protein solution was further purified with a Superdex75 gel filtration column (GE healthcare).

Crystallization, data collection, and structure determination

Purified protein was dialyzed in 10 mM Tris–HCl buffer (pH 8.0) and concentrated to 38 mg/mL. Crystals were obtained in the reservoir solution condition of 16% PEG 3000, 0.1M acetate buffer (pH 4.1), and 25% glycerol at 20°C within 2 days using the sitting drop vapor diffusion method. Collection of X-ray diffraction data was performed at 100 K. The highest resolution data of 1.49 Å was obtained at the SPring-8 BL41XU beamline equipped with an R-AXIS V (Rigaku) detection machine. The diffraction data were processed using the HKL2000 program.6 The crystal belonged to the space group P21 with unit cell parameters of a = 27.95 Å, b = 52.12 Å, c = 33.79 Å, and β = 93.52°. Consideration of the values of VM (VM = 2.0) suggests that this crystal contained one molecule in the asymmetric unit. The parameters of the crystal are summarized in Table I.

Table I. Summary of Data Collection and Refinement Statistics
  • Values in parentheses are for the highest resolution shell.

  • a

    Rmergeequation image, where Ii(hkl) is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I(hkl)〉 its average.

Data collection
Resolution range (Å)50.0–1.49 (1.54–1.49)
Observed reflections57,836
Unique reflections15,890
Data completeness (%)99.9 (99.5)
Redundancy3.6 (3.4)
Rmergea0.039 (0.217)
I〉/〈σ(I)〉32.7 (12.02)
Refinement statistics
Rfactor (%)17.2
Rfree (5.0% total data) (%)19.2
No. protein residues104
No. water molecules114
Average B value (Å2) 
 Main chain atoms15.22
 Side chain atoms17.08
 Water molecules30.97
RMS bond length deviations (Å)0.015
RMS bond angle deviations (°)1.583
Ramachandran plot
Most favored regions (%)96.6
Additional allowed regions (%)3.4
Generously allowed regions (%)0
Disallowed regions (%)0

Molecular replacement was performed with the program Molrep from CCP4 suites7 using the coordinates of spinach chloroplast TRX M (PDB code: 1FB0), which shares 42% sequence identity with ST2123, as a structure model. The initial model was refined by rigid body refinement and restrained refinement using the program Refmac5.8 After a few cycles of refinement, automatic model building and refinement was carried out using the program ARP/wARP.9 Several cycles of manual model rebuilding and refinement were then performed using the programs XtalView10 and Refmac5. One hundred fourteen water molecules were picked up from the FoFc map on the basis of peak heights and distance criteria. The final Rfactor and Rfree were 0.172 and 0.192, respectively. The Ramachandran plot11 deduced from PROCHECK12 shows that 96.6% of residues are included in the most favored regions, and the rest are included in the additionally allowed regions.

Results and discussion

  1. Top of page
  2. Introduction
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Overall structure and active site

The crystal structure of the TRX domain (Val37–Glu140) with the K53E mutant of ST2123 (mstTRX) was successfully determined at 1.49 Å resolution (Fig. 1). It is the first crystal structure of TRX from thermophilic archaea. Though we attempted to determine the crystal structure of wild-type protein (stTRX), we could not completely refine the model to obtain ideal values of Rfactor and Rfree due to the poor quality of the data. The mstTRX forms the typical α/β structure of the TRX fold in which one β-sheet consisting of five β-strands (β1–β5) is surrounded by four α-helices (α1–α4) (Fig. 1). The mstTRX was determined to be the oxidized form in which two cysteine residues at the active site, Cys64 and Cys67, form a disulfide bond (Fig. 1). The overall structure of mstTRX is quite similar to those of the other TRXs (TRXs from Thermus thermophilus, E. coli, Alicyclobacillus acidocaldarius, Anabaena sp., Spinacia oleracea, and Homo sapiens): RMS values of Cα atoms calculated between the structures of mstTRX and other TRXs are from 0.88 to 1.32 Å.

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Figure 1. Ribbon diagram of the structure of mstTRX. The ribbon is colored by gradation from blue (N-terminus) to red (C-terminus). The side chains of the active-site cysteine residues are presented with stick models. The figure was prepared using the PyMOL17 program.

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The tertiary structure of mstTRX is stabilized by some intramolecular interactions: two hydrophobic cores (Core A and Core B), two hydrogen bonds, and four ion pairs. In the hydrophobic cores, Core A is surrounded by short helices of α1 and α3 and Core B by longer helices of α2 and α4 on both sides of the sheet. The two hydrophobic cores contain a number of hydrophobic residues: Core A has seven aliphatic and five aromatic residues, whereas Core B has 13 aliphatic and two aromatic residues. mstTRX also contains two hydrogen bonds by side chain atoms between discrete secondary structure elements. The hydrogen bond of Trp63…Asp92 connects the β2–α2 and β3–α3 loops, and that of Tyr81…Glu131 connects α2 and α4. Furthermore, four ion pairs that connect discrete secondary structure elements are observed in the mstTRX structure. The ion pair of His39…Glu93 connects β1 and the loop between β3 and α3 [Fig. 2(A)]. The ion pairs of Glu75…Lys88 and Asp80…Arg127 connect α2 with β3 and with α4, respectively [Fig. 2(A,B)]. The Asp119…Arg133 ion pair connects β5 and α4 [Fig. 2(B)]. Additionally, Lys53 on the loop between α1 and β2 seems to form an ion pair with Asp114 on the loop between β4 and β5 in the structure of the partly solved wild-type protein (data not shown), although it is disrupted in the structure of mstTRX [Fig. 2(A)].

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Figure 2. Ion pairs of stTRX. A. Side chains of the ion pair residues are presented with yellow stick models. B. Backside view with 150° clockwise rotation from A.

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The active-site motif of TRX, –Trp–Cys–Gly(Ala)–Pro–Cys–, is completely conserved in stTRX [Fig. 3(A)]. The active-site structure is also conserved structurally in mstTRX, which is the first crystal structure of TRX from archaea [Fig. 3(B)]. The RMS values are calculated with active-site residues: the lowest is 0.27 Å with E. coli and the highest is 0.54 Å with Anabaena sp. TRXs. Pro107 near the active site is also conserved, which influences the substrate recognition and structural stability of E. coli TRX and DsbA.13, 14

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Figure 3. A. Sequence alignment of the TRX family. The sequences were aligned with the ClustalW18program, and the figure was produced using ESPript.19The secondary structure and numbering refers to the S. tokodaii sequence. Active-site residues are enclosed by a blue square. B. Stereo diagram of active-site structures from S. tokodaii (black), T. thermophilus (blue), E. coli (pink), A. acidocaldarius (cyan), Anabaena sp. (orange), S. oleracea (green), and H. sapiens (red). The structures are all in the oxidized state except for T. thermophilus and S. oleracea TRXs.

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Characteristic of thermostable protein

S. tokodaii is an organism that grows most optimally at 80°C under low pH conditions.15stTRX was found to be stable at 80°C for 30 min during heat treatment of the purification process and also showed as a stable protein with CD spectra measured at 110°C (data not shown). The increase in the number of ion pairs, which is frequently observed in proteins from thermophilic organisms, was observed on the stTRX surface. Though only two or three ion pairs were observed in the structures of TRXs from E. coli, H. sapiens, and A. acidocaldarius, whose stabilities have been investigated, stTRX possesses five ion pairs that connect the discrete secondary structure elements (ion pair interactions were identified based on the criterion of a distance of 4 Å or less between charged groups16). stTRX has the largest number of ion pairs among TRX proteins whose structures have been reported thus far. The result suggests that an increase in the number of ion pairs would be important for stabilization of stTRX.

Acknowledgements

  1. Top of page
  2. Introduction
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

The synchrotron-radiation experiments were performed at BL41XU in SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (Proposal No. 2003B0827-NL1-np-P3K).

References

  1. Top of page
  2. Introduction
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References