Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1

Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P4₃2₁2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K.

Abstract

Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4₃2₁2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K.

1. Introduction

In protein biosynthesis, maintaining the accurate amino-acid sequence that is important for correct protein structure and function relies on the accurate enzymatic activities of aminoacyl-tRNA synthetases (ARSs). These enzymes catalyze a two-step aminoacylation reaction. An amino acid selected by a cognate ARS is activated by the formation of its aminoacyl-adenylate and the aminoacyl moiety from this adenylate is subsequently transferred to a conserved adenosine residue of the 3′-terminal CCA of the cognate tRNA. The ARS accurately recognizes its cognate amino acid and tRNA and discriminates non-cognate amino acids and tRNAs. The fidelity of this crucial reaction is ensured by the specificity of ARS to the amino acid and its base-specific or tertiary structural interactions with the tRNA (Giegé et al., 1998 ▶; Asahara et al., 1993 ▶).

Tyrosyl-tRNA synthetase (TyrRS) is one of the most researched ARSs and there have been extensive advances in studies of its functional aspects. TyrRS recognizes particular bases and structures of tyrosine tRNA that vary across kingdoms. On the basis of the length of the variable arm, tRNAs are classified into either class I or class II. The class that tyrosine tRNAs belong to differs according to their origin, while the class of other tRNAs is maintained across the three primary domains, namely, bacteria, archaea and eukarya. Bacterial tyrosine tRNA belongs to the class II tRNAs, which are characterized by a long variable arm, while archaeal and eukaryotic tyrosine tRNAs belong to the class I tRNAs, which have short variable arms. On one hand, bacterial TyrRS recognizes the idiosyncratic variable arm by using its flexible C-terminal domain (Yaremchuk et al., 2002 ▶); on the other hand, archaeal and eukaryotic TyrRSs recognize the unique C1–G72 base pairs that are not present in other tRNAs (Kobayashi et al., 2003 ▶; Fechter et al., 2000 ▶, 2001 ▶). Thus, TyrRS provides interesting insights into evolutionary differences in molecular recognition; therefore, understanding its structural information in detail is crucial.

TyrRS from the hyperthermophilic archaeon Aeropyrum pernix K1 is an 81.6 kDa homodimeric protein comprising two 364 amino-acid residue polypeptides. A. pernix TyrRS is strikingly thermostable. It recognizes the C1–G72 base pair of its cognate class I tyrosine tRNA and specifically attaches a tyrosine residue to it (unpublished data). Since a large number of archaeal species survive in extreme habitats resembling those of ancient times, the hyperthermophilic archaeal A. pernix TyrRS will be a good model for studying the molecular-recognition mechanism that has been adapted by TyrRS in two considerably distinct tRNA structures. In this study, the cloning, purification, crystallization and preliminary X-ray diffraction analysis of A. pernix TyrRS are described.

2. Experimental procedures

2.1. Cloning and expression of A. pernix TyrRS

The gene encoding A. pernix TyrRS was isolated from genomic DNA (Kawarabayasi et al., 1999 ▶) using PCR amplification. For PCR, a forward primer (5′-CAT GCC ATG GTC CGC GTG GAT GTT GAG GAG-3′) with an NcoI restriction-enzyme cleavage site (bold) and a reverse primer (5′-CGC GGA TCC GCA TGC CTA TCT CGT AAC CTT ACC TTC TAT-3′) with a BamHI cleavage site (bold) and a termination codon (italicized) were employed. PCR products were inserted into pGEM-T Easy vector (Promega, Madison, WI, USA). DNA fragments digested with NcoI–BamHI were subcloned into corresponding sites of the expression vector pET-28a(+) (Novagen, Madison, WI, USA). None of the histidine tags are present on the expressed A. pernix TyrRS. The expression vectors that encode A. pernix TyrRS were transformed into Escherichia coli BL21-CodonPlus (DE3)-RIL strain (Stratagene, La Jolla, CA, USA). The transformants were cultured in 10 l Luria–Bertani (LB) medium containing 20 µg ml⁻¹ kanamycin at 310 K to an optical density of 0.6 at 600 nm. Protein expression was allowed to proceed for 4 h after induction by the addition of 1 mM isopropylthio-β-d-galactoside (IPTG). Cells were harvested by centrifugation (6000g for 20 min).

2.2. Purification

The harvested cells were suspended in 80 ml lysis buffer (10 mM Tris–HCl pH 8.0, 10 mM KCl, 10 mM MgCl₂, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM β-mercaptoethanol), disrupted by sonication, centrifuged (8000g for 30 min) and the supernatant obtained was heated at 363 K for 1 h to simplify the purification step. Proteins from A. pernix K1 are remarkably thermostable; therefore, cloned A. pernix TyrRS could be easily separated from the heat-denatured host-cell proteins by centrifugation (8000g for 30 min) after the heat treatment. The clarified supernatant was treated with 6 M urea and A. pernix TyrRS was refolded twice in 5 l buffer A (10 mM Tris–HCl pH 8.0, 10 mM KCl, 10 mM MgCl₂, 5 mM β-­mercaptoethanol) to eliminate the effects of misfolding. The dialyzed supernatant was subjected to a Cibacron Blue 3GA affinity column (Sigma, St Louis, MO, USA; 2.5 × 7.0 cm; Li et al., 1999 ▶) equilibrated with buffer A. After washing the column with 300 ml buffer A containing 300 mM NaCl, A. pernix TyrRS was eluted with 100 ml buffer A containing 1.5 M NaCl. Fractions were combined, dialyzed against buffer A and concentrated to 10 mg ml⁻¹ using a Centriprep YM-10 filter (Millipore, Bedford, MA, USA).

2.3. Crystallization and data collection

Initial crystallization conditions were assessed by the hanging-drop vapour-diffusion method using different concentrations of precipitants; namely, ammonium sulfate, polyethylene glycol 6000, 1,6-hexanediol and 2-propanol. A protein solution concentrated to 100 mg ml⁻¹ was used for initial screening. Bipyramid-shaped crystals appeared in the presence of 1.5 M ammonium sulfate under different pH conditions (pH 6.0–8.5) at 293 K. Finally, 1 µl protein solution (10 mg ml⁻¹) and 1 µl reservoir solution consisting of 1.5 M ammonium sulfate and 100 mM Tris–HCl pH 8.0 were mixed and droplets were equilibrated against 400 µl reservoir solution (on 24-well plates; Asahi Techno Glass Co., Tokyo, Japan) at 293 K. Crystals suitable for X-ray analysis were obtained within 3 d (Fig. 1 ▶).

Figure 1.

Typical crystals of A. pernix TyrRS grown using the hanging-drop vapour-diffusion method.

X-ray diffraction experiments were conducted using an R-AXIS VII imaging-plate X-ray detector with a Cu Kα X-ray diffractometer (Rigaku, Japan; λ = 1.5418 Å). After soaking the A. pernix crystals in a cryoprotectant solution (42% xylitol in reservoir solution), they were mounted in a nylon loop (Hampton Research, Aliso Viejo, CA, USA). Subsequently, these crystals were flash-frozen in a nitrogen stream at 100 K. Diffraction data were collected in 0.5° oscillation steps over a range of 100°. The collected data sets were processed using the programs DENZO and SCALEPACK from the HKL2000 package (Otwinowski & Minor, 1997 ▶).

2.4. Aminoacylation assay

The aminoacylation activity was assayed as outlined by Sherman et al. (1992 ▶). Aminoacylation reactions were performed at 338 K in a reaction mixture consisiting of 100 mM HEPES–NaOH pH 8.0, 10 mM KCl, 10 mM MgCl₂, 2 mM ATP, 6.1 µM (¹⁴C)-tyrosine (410 mCi mmol⁻¹) and 8 mg ml⁻¹ total tRNA from A. pernix K1 cells. After pre-incubation for 3 min, the reactions were initiated by adding A. pernix TyrRS (1.0 µg ml⁻¹) to the reaction mixture. The reactions were stopped at constant time intervals by spotting an 8 µl aliquot obtained from 40 µl of the reaction mixture onto Whatman 3MM filter papers (Whatman, Maidstone, Kent, UK) that had been soaked in 5% trichloroacetic acid. After the filter papers had been washed three times (10 min each) with cold 5% trichloroacetic acid, they were dried completely. The radioactivities of the filter papers were measured using a liquid-scintillation counter.

3. Results and discussion

A. pernix TyrRS was cloned and overexpressed in E. coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. The nucleic acids that were tightly bound to A. pernix TyrRS could not be removed using ion-exchange column chromatography (Saldanha et al., 1995 ▶). The high affinity of A. pernix TyrRS to Cibacron Blue 3GA, which can bind enzymes requiring adenylic cofactors such as NAD⁺, NADH and ATP, enabled single-step column chromatography purification. This purification method was extremely rapid and facile compared with the conventional methods, which use several chromatographic steps, and the purified protein was remarkably soluble. The purity of A. pernix TyrRS was detected using denaturing gel electrophoresis and its enzyme activity was determined using an aminoacylation assay. The specific activity of purified A. pernix TyrRS was 487 U mg⁻¹ (1 U of aminoacyl-tRNA synthetase activity was defined as the amount of the enzyme that catalyzes the incorporation of 1 nmol amino acid into aminoacyl-tRNA in 10 min), which was considerably higher than that of human TyrRS (Jia et al., 2003 ▶).

Crystals of A. pernix TyrRS suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to beyond 2.15 Å resolution and belonged to the tetragonal space group P4₁2₁2 or P4₃2₁2, with unit-cell parameters a = b = 66.1, c = 196.2 Å. The native data set contained a total of 898 560 reflections, which reduced to 24 590 unique reflections with 99.7% completeness. The data-collection statistics for A. pernix TyrRS are summarized in Table 1 ▶. Matthews coefficient calculations suggested that the V _M value of the crystal was 2.6 ų Da⁻¹ (Matthews, 1968 ▶), assuming the presence of one A. pernix TyrRS molecule per asymmetric unit; this V _M value corresponded to a solvent content of 52.9%.

Table 1. Data-collection statistics for A. pernix TyrRS.

Values in parentheses refer to the highest resolution shell.

X-ray source	Cu Kα
Wavelength (Å)	1.5418
Space group	P43212
Unit-cell parameters (Å)	a = b = 66.1, c = 196.2
Resolution (Å)	50.0–2.15 (2.23–2.15)
Rmerge (%)	6.5 (33.2)
Completeness (%)	99.7 (99.9)
Average I/σ(I)	55.7 (11.9)
Average redundancy	7.4 (7.3)
Unique reflections	24590 (2392)
Observed reflections	898560

A sequence-homology search conducted using BLAST (Altschul et al., 1997 ▶) for A. pernix TyrRS showed 42% homology to Methanococcus jannaschii TyrRS (PDB code 1j1u; Kobayashi et al., 2003 ▶). The molecular-replacement method using the program MOLREP (Vagin & Teplyakov, 1997 ▶) from the CCP4 package (Collaborative Computational Project, Number 4, 1994 ▶) was employed for structural analysis of A. pernix TyrRS using M. jannaschii TyrRS as the starting model. After rotation, translation and fitting calculations in the resolution range 42.2–3.0 Å, a solution was found when space group P4₃2₁2 was adopted. The crystal contained one molecule per asymmetric unit; therefore, the dimerization axis of A. pernix TyrRS appeared to coincide with the crystallographic axis. Model building and structure determination are currently in progress.