Crystallization and preliminary X-ray analysis of CTP:phosphoethanolamine cytidylyltransferase (ECT) from Saccharomyces cerevisiae

CTP:phosphoethanolamine cytidylyltransferase from S. cerevisiae has been expressed, purified and crystallized.

Abstract

CTP:phosphoethanolamine cytidylyltransferase (ECT) is the enzyme that catalyzes the conversion of phosphoethanolamine to CDP-ethanolamine in the phosphatidylethanolamine-biosynthetic pathway (Kennedy pathway). ECT from Saccharomyces cerevisiae was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 as precipitant. The crystals diffracted X-­rays from a synchrotron-radiation source to 1.88 Å resolution. The space group was assigned as primitive tetragonal, P4₁2₁2 or P4₃2₁2, with unit-cell parameters a = b = 66.3, c = 150.8 Å. The crystals contain one ECT molecule in the asymmetric unit (V _M = 2.2 ų Da⁻¹), with a solvent content of 43%.

1. Introduction

Phosphatidylethanolamine (PE) is a major glycerophospholipid in both prokaryotes and eukaryotes. Two biosynthetic pathways of PE are known in eukaryotes: the CDP-ethanolamine pathway (Kennedy pathway) and decarboxylation of phosphatidylserine. The CDP-ethanolamine pathway, the main pathway of PE biosynthesis (Arthur & Page, 1991 ▶; Tijburg et al., 1989 ▶), involves three enzymatic reactions. Firstly, ethanolamine is phosphorylated at its hydroxyl group to form O-phosphoethanolamine by ethanolamine kinase (EKI). Secondly, CTP is used for cytidylation of O-phosphoethanolamine to synthesize CDP-ethanolamine and a byproduct, pyrophosphate, by CTP:phosphoethanolamine cytidylyltransferase (ECT). Finally, PE is formed from CDP-ethanolamine and diacylglycerol by CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotranferase (EPT). ECT is proposed to be the key regulator of PE synthesis via the CDP-ethanolamine pathway (Sundler, 1975 ▶; Infante & Kinsella, 1979 ▶). ECT consists of two cytidylyltransferase (CT) domains and each CT domain contains a nucleotide-binding motif HXGH (Park et al., 1997 ▶) in its N-­terminal region. Although ECTs from several organisms such as Rattus norvegicus and Chlamydomonus reinhardtii have been well characterized enzymatically, no crystallization of an ECT has been reported so far. Here, we report the crystallization and preliminary X-ray analysis of Saccharomyces cerevisiae ECT, which is encoded by the MUQ1/ECT1 gene (Min-Seok et al., 1996 ▶) and consists of 323 amino-acid residues. In S. cerevisiae ECT, the amino-acid sequence identity between the N- and C-terminal CT domains is 21% and the HXGH motif in the C-terminal CT domain is replaced by HXGD. The most similar protein to ECT with known crystal structures is GCT (sn-glycerol 3-phosphate cytidylyltransferase), an enzyme involved in teichoic acid biogenesis in Gram-positive bacteria. Crystal structures of GCTs from Bacillus subtilis (TagD) and Staphylococcus aureus (TarD), which consist of 129 and 132 amino-acid residues, respectively, comprise a CT domain (Weber et al., 1999 ▶; Pattridge et al., 2003 ▶; Fong et al., 2006 ▶). They are 23 and 24% identical to the N-­terminal CT domain and 23 and 21% identical to the C-terminal CT domain of S. cerevisiae ECT, respectively.

2. Methods and results

2.1. Purification and crystallization

Recombinant S. cerevisiae ECT with an N-terminal 6×His tag and thrombin-cleavage site (MGSSHHHHHHSSGLVPRGSHMLEDQFHM…, where the M in bold is the N-terminal methionine of S. cerevisiae ECT) was overexpressed in Escherichia coli strain BL21(DE3) using pET-15b expression vector (Novagen). E. coli cells were harvested by centrifugation, resuspended in buffer A [25 mM HEPES pH 7.7, 500 mM NaCl, 5%(v/v) glycerol and 2 mM β-mercaptoethanol] and disrupted by sonication. The cell lysate was centrifuged to remove cell debris and the supernatant was applied onto an Ni–NTA agarose (Qiagen) column. The column was washed with buffer A supplemented with 20 mM imidazole and 6×His-tagged ECT was then eluted with buffer A supplemented with 500 mM imidazole. The 6×His tag was cleaved off with thrombin (Amersham Biosciences) and the resulting ECT which has ten extra residues (GSHMLEDQFH) at the N-terminus was separated from 6×His-tagged ECT and 6×His tag using an Ni–NTA agarose column. As the final purification step, gel-filtration chromatography was performed with a HiLoad Superdex 75pg 26/60 column (Amersham Biosciences) equilibrated with buffer B (20 mM MES pH 6.5, 150 mM NaCl and 1 mM TCEP–HCl), which indicated that ECT was monomeric in solution. An Apollo centrifugal ultrafiltration device (Orbital Biosciences) was used to remove NaCl from the protein solution and to concentrate purified ECT to 5.0 mg ml⁻¹.

Crystallization trials were performed by the sitting-drop vapour-diffusion method using Crystal Screens 1 and 2 (Hampton Research). In the presence of 1 mM CTP, small pillar-shaped crystals appeared under condition No. 9 of Crystal Screen 1, in which polyethylene glycol (PEG) 4000 was used as the precipitant. After refinement of the crystallization conditions, crystals suitable for X-ray analysis were obtained with the reservoir solution 24%(w/v) PEG 4000, 100 mM trisodium citrate pH 5.2, 200 mM ammonium acetate. A sitting drop was prepared by mixing 4.0 µl each of protein solution containing 5.0 mg ml⁻¹ ECT, reservoir solution, additive solution (10 mM MgCl₂ and 2 mM CTP dissolved in H₂O) and H₂O to give a total drop volume of 16 µl. In order to reduce the number of crystals in a drop, streak-seeding was applied and drops were equilibrated against 1.0 ml reservoir solution at 278 K. Long pillar-shaped crystals grew in 2 d. Crystals thus obtained are shown in Fig. 1 ▶.

Figure 1.

Crystals of S. cerevisiae ECT in the presence of magnesium ion and CTP with approximate dimensions 1.0 × 0.3 × 0.3 mm. The scale bar corresponds to 0.1 mm.

2.2. X-ray data collection and processing

A crystal of ECT was picked up in a nylon loop (Hampton Research), transferred to a series of cryoprotectant solutions containing 8, 16 and 24%(v/v) glycerol and mounted for flash-cooling at 100 K using a nitrogen stream. The above-mentioned cryoprotectant solutions were prepared by mixing 80%(v/v) glycerol and a solution whose composition was almost identical to the crystallization drop [24%(w/v) PEG 4000, 100 mM trisodium citrate pH 5.2, 200 mM ammonium acetate, 10 mM MgCl₂, 2 mM CTP, 20 mM MES pH 6.5, 1 mM TCEP–HCl].

X-ray diffraction data were collected at beamline NW12 at the Photon Factory Advanced Ring, Tsukuba, Japan with 0.5° oscillations, a crystal-to-detector distance of 180 mm and a wavelength of 1.0000 Å; the crystal diffracted X-rays to 1.88 Å resolution (Fig. 2 ▶). The diffraction data were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997 ▶). The space group was assigned to be P4₁2₁2 or P4₃2₁2 based on the Laue symmetry and the systematic absences. The Matthews coefficient (V _M = 2.2 ų Da⁻¹; Matthews, 1968 ▶) and solvent content (43%) indicated that the crystal contained one ECT molecule per asymmetric unit. The data statistics are summarized in Table 1 ▶.

Figure 2.

An X-ray diffraction image (0.5° oscillation) of a crystal of S. cerevisiae ECT. The edge of the diffraction image corresponds to a resolution of 1.92 Å.

Table 1. Crystal parameters of yeast ECT.

Values in parentheses are for the highest resolution shell.

X-ray source	PF-AR NW12
Wavelength (Å)	1.0000
Space group	P41212 or P43212
Unit-cell parameters (Å)	a = b = 66.3, c = 150.8
Resolution range (Å)	50.0–1.88 (1.91–1.88)
Reflections observed	91691
Unique reflections	28316
Redundancy	3.3 (3.0)
Data completeness (%)	97.4 (92.0)
Mosaicity (°)	0.67
Rmerge†	0.072 (0.291)
〈I〉/〈σ(I)〉	14.6 (3.68)

^† R _merge = , where I_i is the ith intensity measurement of reflection hkl, including symmetry-related reflections, and 〈I〉 is its average.

Structural determination by molecular replacement using the coordinates of GCTs (Weber et al., 1999 ▶; Pattridge et al., 2003 ▶; Fong et al., 2006 ▶) as search models is currently under way.