Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of thymidylate kinase (TTHA1607) from Thermus thermophilus HB8

Thymidylate kinase from T. thermophilus HB8 has been cloned, expressed, purified and crystallized.

Abstract

Nucleotide biosynthesis plays a key role in cell survival and cell proliferation. Thymidylate kinase is an enzyme that catalyses the conversion of dTMP to dTDP using ATP-Mg²⁺ as a phosphoryl-donor group. This enzyme is present at the junction of the de novo and salvage pathways; thus, any inhibitor designed against it will result in cell death. This highlights the importance of this enzyme as a drug target. Thymidylate kinase from the extremely thermophilic organism Thermus thermophilus HB8 has been expressed, purified and crystallized using the microbatch method. The crystals diffracted to a resolution of 1.83 Å and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 39.50, b = 80.29, c = 122.55 Å. Preliminary studies revealed the presence of a dimer in the asymmetric unit with a Matthews coefficient (V _M) of 2.18 ų Da⁻¹.

1. Introduction  

Thermus thermophilus HB8 is a thermophilic, aerobic Gram-negative eubacterium with an optimum growth temperature ranging from 338 to 345 K. It was isolated from thermal vents in the hot springs of Izu, Japan. The proteins present in T. thermophilus are thermally stable compared with their mesophilic homologues (Oshima & Imahori, 1974 ▶; Yoshida & Oshima, 1971 ▶). This makes them industrially important and ideal tools for structural biologists. The present work is based on an enzyme, thymidylate kinase, from T. thermophilus HB8. The enzyme is involved in the nucleotide-biosynthesis pathway which is essential for cell survival and proliferation. Nucleotides (purine and pyrimidine) are biosynthesized by either the de novo pathway or the salvage pathway. The enzyme thymidylate kinase catalyses the reversible conversion of deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) in the presence of ATP-Mg²⁺ which acts as a phosphoryl donor in the reaction. In the de novo pathway of dTMP synthesis, either the phosphorolysis of dUTP or deamination of dCMP yields dUMP, which is further converted to dTMP by thymidylate synthase (Reichard, 1988 ▶). In the salvage pathway of dTMP synthesis, thymidine kinase synthesizes dTMP using thymidine as a substrate (Arnér & Eriksson, 1995 ▶). In later steps thymidylate kinase converts dTMP to dTDP and subsequently dTDP is phoshorylated to dTTP by dNDP kinase for DNA synthesis (Reichard, 1988 ▶). Therefore, thymidylate kinase occurs at the junction of the de novo and salvage pathway and plays a vital role in dTTP synthesis; thus, it is an important drug target for cancer, bacterial and viral diseases (Anderson, 1973 ▶; Neuhard & Nygaard, 1987 ▶).

The three-dimensional crystal structures of thymidylate kinase from various sources have been solved using X-ray crystallography and include structures from Staphylococcus aureus (PDB entry 4f4i; Midwest Center for Structural Genomics, unpublished work), Burkholderia thailandensis (PDB entry 3v9p; Seattle Structural Genomics Center for Infectious Disease, unpublished work) and Ehrlichia chaffeensis (PDB entry 3ld9; Leibly et al., 2011 ▶). The crystal structures of the two native thymidylate kinases from extremely thermophilic organisms, Aquifex aeolicus (PDB entry 2pbr; RIKEN Structural Genomics/Proteomics Initiative, unpublished work) and Sulfolobus tokodaii (PDB entry 2plr; RIKEN Structural Genomics/Proteomics Initiative, unpublished work) have been submitted to the PDB by our group. The crystal structures of thymidylate kinase in complex with various native ligands and inhibitors such as dTDP, dTMP, ADP, AZTMP, TP5A, AppNHp, FLTMP, APPNHp, NH2TMP and (E)-5-(2-bromovinyl)-2′-deoxyuridine-5′-monophos­phate have also been investigated using X-ray crystallography. Most of the enzymes studied have an almost similar active-site topology but display different catalytic activity. The sequence alignment (Fig. 1 ▶) of thymidylate kinase from T. thermophilus (198 amino acids, ∼22 kDa) shows an identity of 23.6% to the enzyme from Homo sapiens, 29% to that from Mycobacterium tuberculosis, 28% to that from Saccharomyces cerevisiae, 20.8% to that from Vaccinia virus and 44.6% to that from A. aeolicus.

Figure 1.

Multiple sequence alignment of thymidylate kinase from T. thermophilus, A. aeolicus, S. cerevisiae, M. tuberculosis, H. sapiens and Vaccinia virus.

Some substrate analogues of thymidylate kinase bind competitively to the enzyme and are converted to the corresponding diphos­phate form and subsequently incorporated into DNA. This incorporation results in chain termination of the newly synthesized DNA strand (Furman et al., 1986 ▶). It has been found that the conversion of the monophosphate substrate analogue to its diphos­phate form is the rate-limiting step which results in the accumulation of the monophosphate form in the cell (Frick et al., 1988 ▶; Fridland et al., 1990 ▶). Thus, there is a need to design better substrate analogues than those currently available. Thymidylate kinase from T. thermophilus may be a suitable system for developing better DNA base analogues for targeting cancer cells. Insights into the reaction mechanism or strategies for drug design based on this enzyme may be applicable to the enzyme from pathogenic microorganisms.

2. Materials and methods  

2.1. Cloning, expression and purification  

The thymidylate kinase gene (TTHA1607) was amplified from T. thermophilus HB8 genome using the primers 5′-ATATCATA­TGCCGGGGCTCTTCCTCACCCTCGA-3′ and 5′-ATATGGATCC­TTATTATGGCAGAAGGGGCCGGAGGTG-3′ by the polymerase chain reaction. The amplified fragment was cloned into the Escherichia coli expression vector pET11a (Novagen, Madison, Wisconsin, USA). The plasmid was transformed into E. coli Rosetta(DE3) (Novagen) cells for protein expression. The transformants were cultured overnight at 310 K in 2 l LB medium containing 100 µg ml⁻¹ ampicillin. The cells were harvested by centrifugation and resuspended in a buffer consisting of 20 mM Tris–HCl pH 8 and 50 mM NaCl, and lysed by sonication. The cell lysate was heated in a dry bath at 343 K for 15 min and the denatured proteins and other cell debris (from the cell lysate) were then separated by centrifugation at 15 000g for 45 min at 277 K. The supernatant was desalted using a Sephadex G-25 (GE Healthcare Biosciences) column pre-equilibrated with 20 mM Tris–HCl pH 8. The desalted fraction was passed through a Sepharose S cation-exchange column (GE Healthcare Biosciences) pre-equilibrated with 20 mM Tris–HCl buffer pH 8 and the protein was eluted using a linear gradient of 0–1.0   NaCl in 20 mM Tris–HCl pH 8. The fraction containing the protein was dialysed against a buffer consisting of 20 mM Tris–HCl, 50 mM NaCl pH 7.4. The dialysed protein sample was further concentrated using a Centricon device (10 kDa molecular weight cutoff). The protein concentration was estimated by measuring the absorbance at 280 nm. The yield of the purified protein was 10 mg per litre of culture.

2.2. Crystallization experiments  

The concentration of the protein solution used for crystallization of the enzyme was 5 mg ml⁻¹ in 20 mM Tris–HCl pH 7.4, 50 mM NaCl. A preliminary screening of the crystallization conditions was performed using Hampton Research Crystal Screen and Crystal Screen 2 by the microbatch-under-oil (Greiner plate) technique. The drops contained 2 µl protein solution and 2 µl crystallization condition. 7 ml of a 1:1 mixture of silicone oil and paraffin oil (1:1) were used in the plate. Diffraction-quality crystals (Fig. 2 ▶) were obtained after 2 weeks in the Crystal Screen condition No. 6 consisting of 0.2 M magnesium chloride hexahydrate, 0.1 M Tris–HCl pH 8.5, 30%(w/v) PEG 4000 at 293 K.

Figure 2.

Crystal used for X-ray data collection.

2.3. Data collection  

A protein crystal was soaked briefly (10 s) in a cryoprotectant solution consisting of mother liquor supplemented with 20%(v/v) glycerol, picked up using a nylon loop and flash-cooled at 100 K in an N₂ cold stream (McFerrin & Snell, 2002 ▶). The diffraction data were collected at 100 K using a MAR 345 imaging-plate detector mounted on a Rigaku UltraX-18 rotating-anode X-ray generator (operated at 40 kV and 80 mA) using the home source available at the Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. The crystal-to-detector distance was 145 mm and the crystal diffracted to a resolution of 1.83 Å. The diffraction data were processed using iMOSFLM (Battye et al., 2011 ▶) and scaled using SCALA (Evans, 2006 ▶) in the CCP4 program suite (Winn et al., 2011 ▶). Details of data collection are summarized in Table 1 ▶.

Table 1. X-ray data-collection statistics.

Values in parentheses are for the last resolution bin.

Crystal habit	Primitive orthorhombic
Wavelength (Å)	1.83
Temperature (K)	100
Space group	P212121
Unit-cell parameters (Å)	a = 39.50, b = 80.29, c = 122.55
Matthews coefficient (Å3 Da−1)	2.18
Solvent content (%)	43.6
No. of molecules in asymmetric unit	2
Resolution range (Å)	40.83–1.83 (1.92–1.83)
No. of observed reflections	251551
No. of unique reflections	33877 (4078)
Completeness (%)	95.5 (80.2)
R merge † (%)	7.2 (57.2)
Multiplicity	7.4 (7.4)
Average 〈I/σ(I)〉	17 (3.3)
Wilson B factor (Å2)	23

^† R _merge = , where I _i(hkl) is the observed intensity and 〈I(hkl)〉 is the average intensity for multiple measurements.

3. Results and discussion  

The thymidylate kinase from T. thermophilus consists of 198 amino acids and has an approximate molecular mass of 22 kDa. A data set was collected from a native protein crystal to 1.83 Å resolution at 100 K. A total of 251 551 reflections are merged into 33 877 unique reflections with an overall R _merge of 7.2% and a completeness of 95.5%. The crystal belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 39.50, b = 80.29, c = 122.55 Å. The Matthews coefficient V _M (Matthews, 1968 ▶) was found to be 2.18 ų Da⁻¹, which suggests the presence of two molecules in the asymmetric unit with a solvent content of 43.6%. The crystal structure of the T. thermophilus thymidylate kinase was solved by molecular replacement using the program MOLREP (Vagin & Teplyakov, 2010 ▶) available in the CCP4 program suite. The three-dimensional atomic coordinates of the thymidylate kinase (PDB entry 2pbr, RIKEN Structural Genomics/Proteomics Initiative, unpublished work) from A. aeolicus were used as a search model for the molecular-replacement calculations (using default parameters). The resultant output model was further subjected to restrained refinement using REFMAC5 (Murshudov et al., 2011 ▶). A total of 5% of the reflections were used for R _free calculations (Brünger, 1992 ▶). The R _work and R _free values of the partially refined model were 43.5 and 46.9%, respectively. Further refinement of the model and the map fitting using Coot (Emsley & Cowtan, 2004 ▶) are in progress.