Crystallization and X-ray data collection of HP0902 from Helicobacter pylori 26695

Two types of His₆-tag-fused HP0902 protein from H. pylori 26695 have been crystallized and X-ray diffraction data were obtained at 1.4 and 2.5 Å resolution.

Abstract

HP0902 from Helicobacter pylori 26695 belongs to the cupin superfamily of proteins, which encompasses proteins with a great diversity in function. In this work, two types of recombinant HP0902 protein were crystallized: one with an N-terminal His₆ tag (^H6HP0902) and the other with a C-terminal His₆ tag (HP0902^H6). The ^H6HP0902 crystal diffracted to 1.40 Å resolution and belonged to space group P2₁, with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å. The HP0902^H6 crystal belonged to space group P4₃2₁2 or P4₁2₁2 and diffracted to 2.5 Å resolution, with unit-cell parameters a = b = 50.4, c = 142.0 Å.

1. Introduction

Infection by the gastric pathogen Helicobacter pylori induces severe gastric disorders, including chronic gastritis, peptic ulcers and stomach cancer. It is reasonable to consider the proteins secreted by H. pylori as potential virulence factors of the bacterium, since they can con­tribute to the gastric inflammation process. For example, the protein VacA has been identified as one of the critical determinants of virulence in H. pylori. VacA is secreted by the bacterium and mediates the pathogenesis of peptic ulceration and gastric cancer in human cells (Isomoto et al., 2010 ▶). HP0902, another protein identified as being secreted by H. pylori (Kim et al., 2002 ▶), has been hypothesized to interact with VacA (Sim, Ahn et al., 2009 ▶). In addition, HP0902 is overexpressed in a mutant strain of H. pylori that lacks the fdxA gene, which regulates resistance to the antibiotic metronidazole (Mukhopadhyay et al., 2003 ▶; Nam et al., 2007 ▶).

Owing to its importance as a human pathogen, the genomes of several strains of H. pylori have been sequenced completely, the first of which was strain 26695 (Tomb et al., 1997 ▶); moreover, structural genomics studies are currently being attempted by several groups around the world (Sim, Lee et al., 2009 ▶). In this context, we initiated a structural study of HP0902, the function of which is un­known. HP0902 is a homodimeric 22 kDa protein. A previous NMR study (Sim, Ahn et al., 2009 ▶; Sim, Lee et al., 2009 ▶), which characterized its secondary structure, implied that this protein belongs to the cupin superfamily. Cupins are ubiquitous proteins that share a highly conserved β-barrel topology and many cupins have been identified as allergens (Mills et al., 2002 ▶; Dunwell et al., 2004 ▶). However, the cupin superfamily includes proteins with a wide variety of functions and sequences and is currently classified into 39 sub­families in the Pfam database (Finn et al., 2010 ▶). Thus, precise analysis of the three-dimensional structure of HP0902 is essential for its functional identification in terms of structural genomics. Since our previous NMR study was unsuccessful in determining its three-dimensional structure, we attempted X-ray crystallography of the protein. Here, we report the crystallization and preliminary X-ray analysis of the HP0902 protein from H. pylori 26695.

2. Materials and methods

2.1. Cloning, expression and purification

The ORF of HP0902 was amplified by PCR using the genomic DNA of H. pylori 26695 (ATCC 700392) as a template. The forward and reverse oligonucleotide primers were 5′-G GAA TTC CAT ATG GAA GTG GTT CAT TTT TTA-3′ and 5′-CCG CCG CTC GAG TTA TTT TTT ACT TAA AGA TAG CCT-3′, respectively, where the NdeI and XhoI restriction-enzyme cleavage sites are shown in bold. The PCR products were digested with NdeI and XhoI and ligated into NdeI/XhoI-digested expression vector pET-15b (Novagen) in order to produce recombinant HP0902 with an N-terminal His₆ tag (^H6HP0902). The resulting construct contained 19 non-native residues at the N-­terminus, including the His₆ tag and the thrombin cleavage site (bold): MGSSHHHHHHSSGLVPRGS. For cloning of recombinant HP0902 with a C-terminal His₆ tag (HP0902^H6), the reverse primer (5′-CCG CCG CTC GAG TTT TTT ACT TAA AGA TAG CCT-3′) did not contain a stop codon and the amplified DNA was inserted into the pET-21a vector (Novagen). This construct contained eight non-native residues at the C-terminus (LEHHHHHH), inclu­ding a His₆ tag that facilitates protein purification. The con­structed plasmids, which were verified via DNA sequencing, were transformed into Escherichia coli strain BL21 (DE3) pLysS. The transformed cells were grown in Luria–Bertani (LB) medium at 310 K. When the A ₆₀₀ reached about 0.6, protein expression was induced by adding IPTG to a final concentration of 1 mM and induction was continued for a further 4 h. Cells were harvested by centrifugation and resuspended in ice-cold buffer A (20 mM Tris–HCl, 10 mM NaCl, 5 mM imidazole pH 8.0). The cells were disrupted by sonication at 277 K and the supernatant was loaded onto a HisTrap FF column (GE Healthcare) pre-equilibrated with buffer A. After extensive washing with the same buffer, the bound protein was eluted using a gradient from 5 to 500 mM imidazole in buffer A. Fractions containing HP0902 were pooled and dialyzed in buffer B (20 mM Tris–HCl, 1 mM DTT pH 8.0) followed by loading onto a HiTrap Q FF column (GE Healthcare) pre-equilibrated with buffer B. The bound protein was eluted using a linear NaCl gradient (10–500 mM) in buffer B. Fractions containing HP0902, which eluted at around 150 mM NaCl, were concentrated to about 2 ml and applied onto a HiLoad 16/60 Superdex 75 column (Pharmacia) that had been equilibrated with the final buffer (10 mM Tris–HCl pH 8.0, 1 mM DTT). The purified solution of each construct was concentrated to around 35 mg ml⁻¹, as estimated via a typical BCA assay, for protein crystallization screening trials. The purified protein was judged to be >95% pure by SDS–PAGE and from the gel-filtration chromatogram (Fig. 1 ▶).

Figure 1.

Purification of ^H6HP0902 and HP0902^H6. (a) Elution profiles of purified ^H6HP0902 (upper panel) and HP0902^H6 (bottom panel) from a gel-filtration column (43 ml void volume and 120 ml total bed volume). The flow rate was 1 ml min⁻¹ and a single peak was observed for the purified protein solution. (b) SDS–PAGE of purified ^H6HP0902 (lane 1, 12 µg; lane 3, 23 µg; lane 5, 58 µg) and HP0902^H6 (lane 2, 17 µg; lane 4, 33 µg; lane 6, 83 µg). Small fractions of dimeric (between 20 and 30 kDa) proteins were detected even under denaturing conditions. Lane M contains molecular-weight markers (labelled in kDa).

2.2. Crystallization

Crystallization of ^H6HP0902 and HP0902^H6 was initially screened by the sitting-drop vapour-diffusion method at 293 K using a Mosquito crystallization robot (TTP LabTech). 0.2 µl protein solution (at 36 mg ml⁻¹ for ^H6HP0902 and 34 mg ml⁻¹ for HP0902^H6) was mixed with 0.2 µl reservoir solution and equilibrated against 70 µl of commercially available reservoir solutions from the Crystal Screen, Crystal Screen 2, Index (Hampton Research), Wizard I and Wizard II (Emerald BioSystems) screening kits. Initial crystals of ^H6HP0902 and HP0902^H6 were formed using Index conditions G1 and G7, respectively. To obtain larger crystals, conditions were manually optimized by changing the PEG concentration and by using the hanging-drop vapour-diffusion method. Drops consisted of 1 µl protein solution mixed with 1 µl reservoir solution and were equilibrated against 1 ml reservoir solution. Well diffracting ^H6HP0902 crystals (Fig. 2 ▶ a) were produced in 3 d at 293 K under the optimal conditions 25%(w/v) polyethylene glycol (PEG) 3350, 0.2 M sodium chloride, 0.1 M Tris–HCl pH 8.5. Diffraction-quality HP0902^H6 crystals (Fig. 2 ▶ b) were produced in 3 d under the condition 25%(w/v) PEG 3350, 0.2 M ammonium acetate, 0.1 M bis-tris pH 6.5.

Figure 2.

Native crystals of (a) ^H6HP0902 and (b) HP0902^H6. The average dimensions of the crystals were approximately 0.1 × 0.1 × 0.05 mm (a) and 0.2 × 0.05 × 0.05 mm (b).

2.3. Data collection

Prior to data collection, single crystals of ^H6HP0902 and HP0902^H6 were picked up in nylon loops and flash-cooled in a nitrogen stream at 100 K. The native crystals were directly mounted under liquid nitrogen for diffraction studies under cryogenic conditions and no ice rings were observed. Additional cryoprotectant was not required as the high PEG content of the crystallization conditions prevented ice-ring formation. In order to find a suitable crystal for data collection, preliminary diffraction tests were performed using a MicroMax-007 HF X-ray generator and an R-AXIS IV⁺⁺ imaging-plate area detector (Rigaku Americas). The final X-ray diffraction data for the native crystals (Fig. 3 ▶) were collected on the BL-17A and BL-1A beamlines of the Photon Factory (PF; Tsukuba, Japan). The collected diffraction data were integrated and scaled using the programs MOSFLM and SCALA from the CCP4 software package (Leslie, 1992 ▶; Evans, 2006 ▶; Winn et al., 2011 ▶).

Figure 3.

Diffraction images of (a) ^H6HP0902 and (b) HP0902^H6 crystals.

3. Results and discussion

As shown in Fig. 2 ▶, prism-shaped and rod-shaped crystals were finally obtained for ^H6HP0902 and HP0902^H6, respectively. The ^H6HP0902 crystal belonged to space group P2₁, with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å, while the HP0902^H6 crystal belonged to space group P4₁2₁2 or P4₃2₁2, with unit-cell parameters a = b = 50.4, c = 142.0 Å (Table 1 ▶). In both crystal forms the unit-cell parameters allowed two molecules of HP0902 in the asymmetric unit, which is consistent with our previous observation that the protein behaves as a homodimer in solution (Sim, Lee et al., 2009 ▶). The Matthews co­efficients calculated for the monoclinic ^H6HP0902 and tetragonal HP0902^H6 crystals were 1.88 ų Da⁻¹ (35% solvent content) and 1.91 ų Da⁻¹ (36% solvent content), respectively (Matthews, 1968 ▶). Self-rotation function analysis (Fig. 4 ▶) of ^H6HP0902 suggested that the two molecules in the asymmetric unit were related by twofold noncrystallographic symmetry (NCS). NCS was not apparent in the tetragonal crystal form (HP0902^H6), which may result from overlap of the twofold NCS axis and one of the crystallographic twofold symmetries.

Table 1. X-ray data-collection statistics for ^H6HP0902 and HP0902^H6 .

Values in parentheses are for the highest resolution bin.

Construct	H6HP0902	HP0902H6
Beamline	BL-17A, PF	BL-1A, PF
Wavelength (Å)	0.98	0.98
Space group	P21	P41212 or P43212
Unit-cell parameters (Å, °)	a = 33.5, b = 78.6, c = 41.4, β = 112.2	a = b = 50.4, c = 142.0
Resolution range (Å)	71–1.40	71–2.50
No. of observations	250905	31274
No. of unique reflections	38998	6158
Rmerge†	0.054 (0.362)	0.042 (0.310)
Multiplicity	6.4	5.1
Completeness (%)	99.8	98.2
Mean I/σ(I)	15.8 (3.6)	39.9 (5.09)

^† R _merge = .

Figure 4.

Self-rotation plots for the data sets of (a) ^H6HP0902 and (b) HP0902^H6. Self-rotation maps were generated using the MOLREP program (Vagin & Teplyakov, 2010 ▶) and contoured at the χ = 180° section for the monoclinic (a) and tetragonal (b) crystal forms.

In our initial attempts to crystallize HP0902 without a His₆ tag, needle-shaped crystals were obtained for ^ΔH6HP0902, which was formed by cleavage of the N-terminal His₆ tag from ^H6HP0902, but attempts to improve the crystal quality failed (data not shown). However, despite being fused to a His₆ tag, both the ^H6HP0902 and HP0902^H6 constructs readily crystallized; the position of the His₆ tag probably affected the crystal-packing process in favourable ways, thereby changing the shape of the crystals (which belong to different space groups and have different unit-cell parameters; Table 1 ▶). In particular, the crystal of the N-terminally His₆-tag-fused construct ^H6HP0902 diffracted to a high resolution of 1.40 Å. However, in many cupin-family proteins the N-terminus plays a critical role in intermolecular contacts for dimerization and these may have been affected by the N-terminal His₆ tag of the ^H6HP0902 construct. Thus, we additionally collected diffraction data from the HP0902^H6 crystal, in which a His₆ tag was fused at the C-terminus. Since the HP0902^H6 crystal diffracted to 2.5 Å resolution, which is sufficient for diffraction analysis, the two crystal structures should supplement and validate each other, thereby providing indubitable information on the native intact protein structure. Structure determination using molecular replacement is under way. The high-resolution crystal structure of HP0902 will be valuable in elucidating the function of this interesting protein secreted by the clinically important pathogen H. pylori.