Purification, crystallization and preliminary crystallographic analysis of the vacuole-type ATPase subunit E from Pyrococcus horikoshii OT3

The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 Å resolution with 98.8% completeness and an R_merge of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 Å, and is most likely to contain one molecule per asymmetric unit.

1. Introduction

The vacuolar ATPases (V-ATPases) are found in many organisms and are involved in various physiological processes (Nishi & Forgac, 2002). They play major roles in endomembrane and plasma-membrane proton transport in eukaryotes, are present in the membranes of yeast vacuoles, clathrin-coated vesicles, lysosomes and plant vacuoles, and pump protons into vesicles (Yokoyama et al., 2000). In eukaryotic cells, V-ATPases translocate protons across the membrane by consuming ATP. Eukaryotic V-ATPases account for various cell functions including the acidification of intracellular compartments, renal acidification, bone resorption and tumour metastasis (Yoshida et al., 2001). V-ATPases and the F-type ATP synthases (F-ATPases) are evolutionarily related and share the rotary method of coupling ATP synthesis/hydrolysis and proton translocation across the membrane (Yoshida et al., 2001; Imamura et al., 2003; Yokoyama, Nakano et al., 2003). Like F-ATPases, V-ATPases are oligomeric complexes composed of a water-soluble set of subunits (V₁) and membrane-integral subunits (V₀). However, the subunit composition and structure of V-ATPases are reported to be significantly different from those of F-ATPases (Yokoyama, Nakano et al., 2003).

V-ATPases are also found in some eubacteria (prokaryotic V-­ATPases) and archaea (Yokoyama et al., 1998). The V-ATPase from Pyrococcus horikoshii, which lacks F-ATPase, is responsible for ATP synthesis in this archaeon (Yokoyama et al., 2000). It is composed of nine different subunits, G-I-L-E-C-F-A-B-D, which are proteins with molecular sizes of 13, 71, 8, 20, 35, 12, 64, 54 and 25 kDa, respectively (Yokoyama, Nagata et al., 2003). The V₁ region consists of four subunits with stoichiometry A₃B₃D₁F₁ (Yokoyama et al., 1990), whereas the V₀ region, which is involved in proton-channel activity, is composed of five different subunits, two typical membrane-spanning subunits I and L and three hydrophilic subunits E, G and C. Understanding the structural and functional role of the V-ATPase subunits is essential as they may provide the uniqueness that distinguishes them from the F-ATPases. The precise arrangement of the subunits in the V-ATPase remains an important unclarified issue. Recently, the structure of V-ATPase subunit C from Thermus thermophilus was determined using X-ray crystallography (Iwata et al., 2004) and a solution study of the yeast enzyme (Armbrüster et al., 2004) has also been reported. Three-dimensional structures of the other V-ATPase subunits have not yet been reported. Here, we report the purification and preliminary crystallographic study of V-ATPase subunit E from P. horikoshii OT3. V-ATPase subunit E was predicted to be a highly hydrophobic α-helical protein and one of the candidates for the F₁ γ-subunit homologue (Grüber et al., 2000; Chaban et al., 2002). Biochemical studies strongly suggest that the E subunit is a stator subunit rather than a rotor subunit (Arata et al., 2002; Yokoyama, Nagata et al., 2003). The three-dimensional structure of this subunit would help in understanding the molecular mechanism of the V-ATPase. The V-ATPase subunit E from P. horikoshii OT3 has a molecular weight of 22.88 kDa and consists of 198 amino-acid residues.

2. Experimental

2.1. Protein expression and purification

The plasmid encoding the V-ATPase subunit E was digested with NdeI and BglII and the fragment was inserted into the expression vector pET-11a (Novagen) linearized with NdeI and BamHI. Escherichia coli BL21 Codon Plus (DE3)-RIL cells were transformed with the recombinant plasmid and grown at 310 K in Luria–Bertani medium containing 50 µg ml⁻¹ ampicillin for 20 h. The cells were harvested by centrifugation at 4500g for 5 min at 277 K and were subsequently suspended in 20 mM Tris–HCl pH 8.0 containing 0.5 M NaCl and 5 mM 2-mercaptoethanol; they were then disrupted by sonication and heated at 363 K for 11.5 min. The cell debris and denaturated protein were removed by centrifugation at 18 000g for 30 min. The supernatant solution was used as the crude extract for purification. The crude extract was desalted using a HiPrep 26/10 desalting column (Amersham Biosciences) and applied onto a Super Q Toyopearl 650 M (Tosoh) column equilibrated with 20 mM Tris–HCl pH 8.0 (buffer A). After elution with a linear gradient of 0–0.3 M NaCl, the fraction containing V-ATPase subunit E was desalted using a HiPrep 26/10 desalting column (Amersham Biosciences) with buffer A. The sample was loaded onto a Resource Q column (Amersham Biosciences) equilibrated with buffer A. After elution with a linear gradient of 0–0.3 M NaCl, the fraction containing the V-­ATPase subunit E was desalted using a HiPrep 26/10 desalting column with 10 mM sodium phosphate pH 7.0. The sample was then applied onto a Bio-Scale CHT-5-I column (Bio-Rad) equilibrated with 10 mM sodium phosphate pH 7.0 and eluted with a linear gradient of 10–300 mM sodium phosphate pH 7.0. The sample was concentrated by ultrafiltration (Vivaspin, 10 kDa cutoff) and loaded onto a HiLoad 16/60 Superdex 200 prep-grade column (Amersham Biosciences) equilibrated with buffer A containing 0.2 M NaCl. The homogeneity and identity of the purified sample were assessed by SDS–PAGE (Laemmli, 1970) and N-terminal sequence analysis. Finally, the purified V-ATPase subunit E was concentrated by ultrafiltration to 25.43 mg ml⁻¹ in buffer A containing 0.2 M NaCl and the protein stock solution was stored at 203 K.

2.2. Crystallization

Crystals of V-ATPase subunit E were obtained by the microbatch method using Nunc HLA plates (Nalge Nunc International). Initial crystallization conditions were obtained using the TERA (automatic crystallization) system from 144 independent conditions (Sugahara & Miyano, 2002). After optimization, each crystallization drop was prepared by mixing equal volumes (1.0 µl) of precipitant solution [27.5%(w/v) PEG 4000, 0.1 M CHES pH 8.5] and protein stock solution (§2.1). The crystallization drop was then overlaid with a 1:1 mixture of silicon and paraffin oils, allowing slow evaporation of water in the drop, and stored at 296 K.

2.3. X-ray data collection and analysis

Initial tests for the diffraction quality of the crystals were performed using a Rigaku R-AXIS VII image-plate area detector and Cu Kα X-rays (Rigaku, Japan). A crystal of V-ATPase subunit E from P. horikoshii was flash-cooled in a cryoprotectant solution consisting 27.5%(w/v) PEG 4000, 0.1 M CHES and 30%(v/v) glycerol pH 8.5 at 100 K. X-ray diffraction data were collected to 1.85 Å from the vitrified crystals at SPring-8 beamline BL26B1 using a Rigaku R-AXIS V image-plate area detector. 140 frames were collected, with 1° oscillation and 30 s exposure time per frame. The wavelength of the synchrotron radiation was 1.000 Å and the distance between the crystal and detector was 250 mm. Data were indexed, integrated and scaled using HKL2000 (Otwinowski & Minor, 1997).

3. Results and discussion

Well diffracting crystals of the V-ATPase subunit E appeared about 20–22 d after setup and grew to approximate dimensions of 0.5 × 0.1 × 0.01 mm after six weeks (Fig. 1). A complete data set to 1.85 Å resolution was collected at SPring-8 (Japan) beamline BL26B1 and the data-collection statistics are reported in Table 1. A total of 90 376 measured reflections in the resolution range 30–1.85 Å were merged into 19 506 unique reflections with an R_merge of 6.5%. The native crystal belongs to the orthorhombic space group P2₁2₁2₁. The asymmetric unit is most likely to contain a single chain of the V-­ATPase subunit E; the crystal volume per unit molecular weight, V_M, was calculated to be 2.5 ų Da⁻¹, corresponding to a solvent content of 50.4% (Matthews, 1968). Crystals of the selenomethionine-labelled protein have been obtained under the same conditions as the native protein and appear to have similar diffraction properties. The structure will be determined using the MAD method (Hendrickson et al., 1990).

Table 1
X-ray data-collection statistics for the V-ATPase subunit E

Values in parentheses are for data in the highest resolution shell.

Space group	P212121
Unit-cell parameters (Å)	a = 52.196, b = 55.317, c = 77.481
Wavelength (Å)	1.000
Resolution range (Å)	30–1.85 (1.92–1.85)
Total No. observed reflections	90376
Total No. unique reflections	19506
Completeness (%)	98.8 (98.6)
Redundancy	4.7(4.8)
Mean I/σ(I)	11.6 (5.8)
Rmerge† (%)	6.5 (27.7)

†R_merge = , where I_j(hkl) and 〈I(hkl)〉 are the observed intensity of measurement j and the mean intensity for the reflection with indices hkl, respectively.

Figure 1 A crystal of V-ATPase subunit E from P. horikoshii OT3. The crystal has approximate dimensions of 0.5 × 0.1 × 0.01 mm.