Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNA^Ile2 and ATP

A. fulgidus TiaS was cocrystallized with tRNA^Ile2 and ATP and X-ray diffraction data were collected to 2.9 Å resolution using a synchrotron-radiation source.

Abstract

The cytidine at the first anticodon position of archaeal tRNA^Ile2, which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm²C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA^Ile-agm²C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA^Ile2 was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA^Ile2 and ATP by the vapour-diffusion method. The crystals of the TiaS–tRNA^Ile2–ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3₂21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS–tRNA^Ile2–ATP complex, with a Matthews coefficient of 2.8 ų Da⁻¹ and a solvent content of 61%.

1. Introduction

Post-transcriptional modifications at the first position (wobble position or position 34) of the tRNA anticodon play an important role in the precise decoding of the genetic code (Björk, 1995 ▶; Suzuki, 2005 ▶; Yokoyama & Nishimura, 1995 ▶). In most archaea, the wobble cytidine (C34) of the AUA codon-reading isoleucine tRNA (tRNA^Ile2) is modified to 2-agmatinylcytidine (agm²C or agmatidine), an agmatine-conjugated cytidine derivative (Ikeuchi et al., 2010 ▶; Mandal et al., 2010 ▶). The precursor form of tRNA^Ile2 with unmodified C34 is mis-aminoacylated by methionyl-tRNA synthetase and deciphers the methionine AUG codon (Ikeuchi et al., 2010 ▶; Köhrer et al., 2008 ▶), because tRNA^Ile2 bears the CAU anticodon sequence that also appears in tRNA^Met. In contrast, tRNA^Ile2 with the agm²C modification precisely deciphers the isoleucine AUA codon instead of AUG (Ikeuchi et al., 2010 ▶). In addition, mature tRNA^Ile2 is charged with isoleucine by isoleucyl-tRNA synthetase in vitro owing to the presence of the agm²C modification (Köhrer et al., 2008 ▶). Thus, the agm²C modification switches the codon specificity from AUG to AUA and enables tRNA^Ile2 to be correctly aminoacylated with isoleucine. Therefore, the agm²C modification is crucial for accurate decoding of the genetic code.

The formation of agm²C is catalyzed by tRNA^Ile-agm²C synthetase (TiaS) using ATP and agmatine as substrates (Ikeuchi et al., 2010 ▶). In its primary structure, TiaS has three conserved motifs: DUF1743, an OB-fold and a zinc finger-like motif (Ikeuchi et al., 2010 ▶). A mutational analysis of conserved residues in each motif revealed that all of these motifs are essential for agm²C formation (Ikeuchi et al., 2010 ▶). As observed in many RNA-binding proteins (Krishna et al., 2003 ▶; Schmitt et al., 1998 ▶), both the OB-fold and zinc finger-like motifs in TiaS would be involved in tRNA recognition. TiaS requires ATP during agm²C formation, but there is no clear ATP-binding motif in its primary structure. In addition to the three conserved motifs, TiaS has several conserved residues in the N-terminal region upstream of DUF1743. Thus, the uncharacterized DUF1743 and/or the N-­terminal region of TiaS might serve as an ATP-binding motif for agm²C formation. Several crystal structures of ATP-utilizing tRNA-modification enzymes have been reported (Chimnaronk et al., 2009 ▶; Nakanishi et al., 2009 ▶; Numata et al., 2006 ▶; Waterman et al., 2006 ▶), but TiaS lacks sequence similarity to these enzymes. Therefore, the ATP-binding mode and the catalytic mechanism of agm²C formation by TiaS remain to be elucidated. The structural analysis of the complex containing TiaS and its substrates is indispensable for revealing the mechanism of agm²C formation. In the present paper, we report the crystallization and preliminary X-ray diffraction analysis of Archaeo­globus fulgidus TiaS in complex with tRNA^Ile2 and ATP.

2. Materials and methods

2.1. Protein preparation

The DNA fragment encoding A. fulgidus TiaS was produced by PCR amplification and cloned into the expression vector pET15b (Novagen). Recombinant TiaS with an N-terminal His tag was produced in Escherichia coli strain C41 (DE3) (Lucigen). After sonication of the E. coli cells in 20 mM Tris–HCl pH 8.0 containing 300 mM NaCl, 10 mM imidazole, 7 mM β-mercaptoethanol, 1 mM PMSF and 1 mM benzamidine, the clarified lysate was incubated at 338 K for 20 min. The heat-treated lysate was centrifuged at 8000 rev min⁻¹ for 30 min and the supernatant was loaded onto an Ni–NTA (Qiagen) column equilibrated with buffer A (20 mM Tris–HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 7 mM β-mercapto­ethanol). TiaS was eluted with buffer B (buffer A containing 250 mM imidazole). The eluate from the Ni–NTA column was dialyzed against buffer C (20 mM Tris–HCl pH 8.0, 200 mM NaCl, 20 mM imidazole pH 7.0, 7 mM β-mercaptoethanol). The protein solution was then loaded onto a HiTrap Heparin column (16 × 25 mm; GE Healthcare) previously equilibrated with buffer C. A linear gradient from 200 to 1000 mM NaCl was developed in buffer C. The fractions enriched in the target protein were combined and dialyzed against buffer D (20 mM Tris–HCl pH 8.0, 500 mM NaCl, 7 mM β-mercaptoethanol). The protein solution was further purified on a Superdex 200 column (1.6 × 60 cm; GE Healthcare) previously equilibrated with buffer D. The fractions enriched in TiaS were combined and concentrated to 1.0–1.9 mg ml⁻¹. Selenomethionine-labelled (SeMet-labelled) TiaS was purified using the same procedure as was used for the native protein.

2.2. tRNA preparation

The gene encoding A. fulgidus tRNA^Ile2 was PCR-amplified and cloned into the pUC18 vector. This plasmid was digested with the FokI restriction enzyme and the digest was used as a template for in vitro transcription with T7 RNA polymerase. The transcribed tRNA^Ile2 was purified under denaturing conditions by polyacrylamide gel electrophoresis and was eluted from the gel pieces. After ethanol precipitation, tRNA^Ile2 was dissolved in water and then passed through a PD-10 column (GE Healthcare) to completely remove the residual urea. tRNA^Ile2 was further precipitated with ethanol, dissolved in water and stored at 193 K.

2.3. Crystallization

The purified tRNA^Ile2 in 10 mM Na HEPES pH 7.5 and 5 mM MgCl₂ was denatured by heat treatment at 343 K for 10 min and was then refolded by gradual cooling of the solution to 298 K. TiaS was mixed with tRNA^Ile2 in a 1:1.2 molar ratio of TiaS:tRNA^Ile2 at a final protein concentration of 0.9–1.8 mg ml⁻¹ and the mixture was incubated at 310 K in the presence of 5 mM MgCl₂, 1 mM DTT and 1 mM ATP. After the sample solution had been placed on ice for 15 min, crystallization conditions were screened by the sitting-drop vapour-diffusion method at 293 K. Sitting drops were prepared by mixing 1 µl reservoir solution with 1 µl sample solution and were equilibrated against 100 µl reservoir solution. Crystals grew using a reservoir solution consisting of 25.5% PEG 4000, 0.17 M sodium acetate, 0.085 M Tris–HCl pH 8.5, 15% glycerol. We subsequently optimized the crystallization conditions by the hanging-drop vapour-diffusion method. Hanging drops were prepared by mixing 2 µl reservoir solution with 2 µl sample solution and were equilibrated against 500 µl reservoir solution. Consequently, crystals grew to dimensions sufficient for X-ray diffraction analysis using a reservoir solution consisting of 26.5–28% PEG 4000, 0.17 M sodium acetate, 0.085 M Tris–HCl pH 8.5, 15% glycerol. Under the optimized reservoir conditions, SeMet-labelled TiaS was also cocrystallized with tRNA^Ile2 and ATP by the hanging-drop vapour-diffusion method. To determine whether the crystals contained the TiaS–tRNA^Ile2 complex, the crystals were harvested, washed well, dissolved in Milli-Q water and examined by SDS–PAGE and denaturing PAGE.

2.4. X-ray data collection

The TiaS–tRNA^Ile2–ATP complex crystals were polycrystals. For X-ray data collection, the polycrystals were carefully separated into single crystals using a needle. The separated single crystals were mounted in a cryoloop and then directly flash-cooled in a nitrogen stream at 95 K. X-ray diffraction data were collected on beamline BL-17A of KEK (Ibaraki, Japan) using an ADSC Q270 CCD detector. Diffraction data were integrated and scaled with the program HKL-2000 (Otwinowski & Minor, 1997 ▶). The processing statistics are summarized in Table 1 ▶.

Table 1. Data-collection statistics.

Values in parentheses are for the last shell.

Wavelength (Å)	0.97898
Space group	P3221
Unit-cell parameters (Å, °)	a = b = 131.1, c = 86.6, α = β = 90, γ = 120
Resolution (Å)	50–2.90 (2.95–2.90)
Measured reflections	295793
Unique reflections	19261
Multiplicity	15.4 (8.2)
Completeness (%)	99.9 (99.9)
〈I/σ(I)〉	29.3 (2.7)
Rmerge†	0.100 (0.407)

^† R _merge = , where I _i(hkl) is the observed intensity and 〈I(hkl)〉 is the average intensity over symmetry-equivalent measurements.

3. Results and discussion

The TiaS protein, with a His tag attached to its N-terminus, was produced in E. coli strain C41 (DE3) using the pET15b expression vector and was purified to homogeneity through several chromatographic steps. The amount of recombinant TiaS obtained by this procedure was 1.0 mg from 8 l culture. tRNA^Ile2 was prepared by in vitro transcription using T7 RNA polymerase. The purified TiaS and tRNA^Ile2 were mixed in a 1:1.2 molar ratio and the mixture was used for crystallization screening by the sitting-drop vapour-diffusion method at 293 K. The crystals were obtained under conditions consisting of 25.5% PEG 4000, 0.17 M sodium acetate, 0.085 M Tris–HCl pH 8.5, 15% glycerol. Electrophoretic analyses of the obtained crystals by SDS–PAGE and denaturing PAGE demonstrated that the crystals contained both TiaS and tRNA^Ile2 (data not shown). We subsequently optimized the crystallization conditions by the hanging-drop vapour-diffusion method and obtained complex crystals suitable for X-ray diffraction experiments using the reservoir conditions of 26.5–28% PEG 4000, 0.17 M sodium acetate, 0.085 M Tris–HCl pH 8.5, 15% glycerol. The complex crystals grew to maximum dimensions of 40 × 20 × 20 µm within a week (Fig. 1 ▶). We also obtained complex crystals containing SeMet-labelled TiaS, tRNA^Ile2 and ATP by the hanging-drop vapour-diffusion method. The complex crystals diffracted to 2.9 Å resolution (Fig. 2 ▶) and belonged to the primitive hexagonal space group P3₂21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. On the basis of the molecular mass of the recombinant TiaS (50 kDa) and tRNA^Ile2 (25 kDa), the complex crystals are expected to contain one TiaS–tRNA^Ile2 complex per asymmetric unit, which corresponds to a solvent content of 61% and a Matthews coefficient of 2.8 ų Da⁻¹. Quite recently, we have succeeded in determining the TiaS–tRNA^Ile2–ATP ternary complex structure by the single-wavelength anomalous dispersion (SAD) method using the SeMet-labelled crystals. In the SAD experimental electron-density map we observed clear density for ATP in addition to that for the TiaS protein and tRNA^Ile2, demonstrating that the crystals definitely contain an ATP molecule and shedding light on the structural aspects of the mechanism of agm²C formation. The structural analysis of the TiaS–tRNA^Ile2–ATP ternary complex will be discussed elsewhere in the future.

Figure 1.

Crystals of the TiaS–tRNA^Ile2–ATP complex.

Figure 2.

Diffraction pattern of a TiaS–tRNA^Ile2–ATP complex crystal.