Crystallization and preliminary crystallographic studies of Helicobacter pylori arginase

In this study, H. pylori arginase was purified and crystallized in complex with Mn²⁺ and a diffraction data set was collected to 2.2 Å resolution.

Abstract

Helicobacter pylori arginase is an important factor in evasion of the host’s immune system and contributes to persistent infection by this bacterium. It is unique in many aspects compared with other arginases: for example, it has optimal activity with Co²⁺ as a cofactor rather than Mn²⁺ and has strongest activity at acidic pH instead of alkaline pH. In this study, H. pylori arginase was purified and crystallized in complex with Mn²⁺ and a diffraction data set was collected to 2.2 Å resolution. The crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 94.69, b = 102.24, c = 148.61 Å.

1. Introduction

Helicobacter pylori infects more than 50% of the world population; it is the cause of gastritis and peptic ulcers and a risk factor for gastric cancer (Kusters et al., 2006 ▶). It usually persists for the life of the host and is not eradicated despite a vigorous immune response (Suerbaum & Michetti, 2002 ▶). The persistent infection by this bacterium results from immune evasion by several effectors, such as the virulence factors CagA and VacA (Cooke et al., 2005 ▶). Arginase is another protein that has recently been identified to show immunosuppressant properties and is probably involved in the bacterium’s evasion of the host’s immune system (Baldari et al., 2005 ▶). It is a urea-cycle enzyme that catalyzes the hydrolysis of arginine to yield ornithine and urea (Muszyńska et al., 1972 ▶) and is present in almost all known life forms, playing a crucial role in nitrogen metabolism. H. pylori arginase competes with host inducible nitric oxide synthase for the common substrate l-arginine; this reduces the synthesis of NO, an important component of innate immunity and an effective antimicrobial agent that is able to kill invading pathogens directly (Bussière et al., 2005 ▶). Additionally, H. pylori arginase is able to inhibit human T-cell proliferation and T-cell CD3ζ expression and thus efficiently reduces the host cellular immune response (Zabaleta et al., 2004 ▶). Also, its product urea is utilized by urease to generate ammonia and carbon dioxide, which helps to neutralize the acidic environment of the stomach and thus facilitate colonization by the bacterium (McGee et al., 1999 ▶).

Two arginase isozymes have been identified in eukaryotes: arginase I and II. The former is highly expressed in liver and plays a key role in the urea cycle, while the latter is a mitochondrial enzyme that is involved in l-arginine homeostasis (Kepka-Lenhart et al., 2008 ▶). Arginases from many bacteria have also been identified, such as those from Bacillus caldovelox (Bewley et al., 1999 ▶) and Streptomyces clavuligerus (Elkins et al., 2002 ▶). The activity and catalytic mechanism of arginases from eukaryotes and some bacteria have been well characterized. These enzymes show highest activity in the presence of manganese and have an optimum pH of 9.0–11.0 (Jenkinson et al., 1996 ▶; Bussière et al., 2005 ▶; Kanyo et al., 1996 ▶). However, H. pylori arginase is only distantly related to arginases of known structure (28% sequence identity to the closest homologue B. caldovelox arginase; PDB entry 1cev; Bewley et al., 1999 ▶) and is unique in the following aspects. (i) It shows optimal activity with Co²⁺ as a cofactor rather than Mn²⁺ (McGee et al., 2004 ▶). (ii) It shows strongest activity at acidic pH (pH 6.1) instead of alkaline pH (McGee et al., 2004 ▶), which may be an adaptation to the acidic colonization environment of H. pylori. (iii) It contains an insertion corresponding to residues 153–165 which was not found in other arginases and which has been proposed to interact with the membrane since this enzyme is cell-envelope associated (Mendz et al., 1998 ▶). (iv) It is extremely sensitive to reducing agents such as dithiothreitol and β-­mercaptoethanol and its activity can be inhibited by small doses of reducing agent (McGee et al., 2004 ▶). (v) It has been identified to exist as a monomer in solution (work to be published), in contrast to typical trimeric or hexameric (Bewley et al., 1999 ▶) complexes, although a human argin­ase has been reported to be active as a monomer (Aguirre & Kasche, 1983 ▶).

To identify the role of H. pylori in infection and study its unique biochemical and enzymatic properties, we intend to determine the structure and enzymatic mechanism of H. pylori arginase. Here, we report the crystallization, diffraction data collection and preliminary crystallographic studies of arginase from H. pylori.

2. Materials and methods

2.1. Cloning, expression and purification

The rocF gene (NCBI Gene ID 899897) encoding H. pylori argin­ase was amplified from the genome of H. pylori 26695 and cloned into an expression vector derived from the pET-22b(+) plasmid (Novagen) and placed between NdeI and XhoI restriction sites. The H. pylori arginase C-terminus was fused to a His₆ tag (LEHHHHHH) in order to facilitate purification. The insert was sequenced and found to be in complete agreement with the expected sequence.

Escherichia coli BL21 (DE3) pLysS strain (Invitrogen) transformed with plasmid pET22b-rocF was grown at 310 K in 2 l Luria–Bertani medium supplemented with ampicillin (100 mg ml⁻¹) until the optical density at 600 nm (OD₆₀₀) reached 0.6. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM to induce expression of the recombinant protein. Cell growth was continued at 310 K for 3 h. The cells were harvested by centrifugation and the bacterial pellets were resuspended in lysis buffer (20 mM phosphate buffer pH 8.0, 300 mM NaCl and 10 mM imidazole) and disrupted by ultrasonication. Insoluble cellular material was removed by centrifugation. The supernatant was loaded onto a nickel–nitrilotriacetic acid agarose (Ni–NTA; Novagen) column previously equilibrated with lysis buffer. The column was washed with wash buffer (20 mM phosphate buffer pH 8.0, 300 mM NaCl and 20 mM imidazole). His-tagged H. pylori RocF was then eluted using washing buffer (20 mM phosphate buffer pH 8.0, 300 mM NaCl and 250 mM imidazole). The eluted protein was concentrated to about 1 ml by ultrafiltration (Millipore) and loaded onto a Superdex 200 HiLoad 16/60 column (GE Healthcare) pre­viously equilibrated with 20 mM Tris–HCl pH 7.5, 150 mM NaCl. The peak corresponding to the H. pylori arginase monomer was collected; the protein was concentrated to about 80 mg ml⁻¹ and stored at 277 K for further crystallization experiments. Protein concentrations were determined from the absorbance at 280 nm, assuming an A ₂₈₀ of 0.543 for a 1.0 mg ml⁻¹ solution.

2.2. Crystallization

Initial crystallization screening for the H. pylori arginase used Crystal Screen, Index, PEG/Ion and PEG/Ion 2 from Hampton Research. Crystallization experiments were set up via the hanging-drop vapour-diffusion technique at 293 K. Each drop was formed by mixing equal volumes (1 µl) of protein solution (80 mg ml⁻¹ arginase in 20 mM Tris–HCl pH 7.5, 150 mM NaCl) and reservoir solution and was allowed to equilibrate against 420 µl reservoir solution. Conditions giving positive hits were optimized by varying the type and the concentration of precipitants, salts, buffers, organic compounds and additives (Additive Screen, Hampton Research).

2.3. Data collection and processing

Prior to data collection, the H. pylori arginase crystals were briefly soaked (for about 15 s) in cryoprotectant, which corresponded to the mother liquor with the addition of 15%(v/v) glycerol. Crystals were then mounted for X-ray data collection using CryoLoops (Hampton Research) and flash-cooled in a nitrogen-gas stream at 95 K. A native data set to 2.2 Å resolution was collected at a wavelength of 1.0000 Å on beamline 17U of the Shanghai Synchrotron Radiation Facility using a CCD detector. We collected 180 images with 1° oscillation per image. MOSFLM (v.7.0.4; Leslie, 2006 ▶) and SCALA (v.6.0) from the CCP4 program suite (v.6.0.2; Winn et al., 2011 ▶) were used for indexing, integration and scaling of the diffraction data set.

3. Results

Crystals were initially obtained from Hampton Research Index screen condition No. 65 [0.1 M ammonium acetate, 0.1 M Bis-Tris pH 5.5, 17%(w/v) PEG 10 000] and PEG/Ion screen condition No. 3 [0.2 M ammonium fluoride, 20%(w/v) PEG 3350 pH 6.2]. The optimized crystallization condition consisted of 25% polyethylene glycol 3350, 100 mM Bis-Tris pH 5.5, 1 mM MnCl₂, 15 mM guanidine hydrochloride using hanging-drop vapour diffusion; equal volumes (1 µl) of protein solution and reservoir solution and 0.3 µl 30%(w/v) 1,5-diaminopentane were mixed at room temperature. Crystals appeared overnight and grew to dimensions of about 0.5 × 0.1 × 0.06 mm in a few days (Fig. 1 ▶). Data-collection statistics for the native crystal are shown in Table 1 ▶. The crystal diffracted to a resolution of 2.2 Å and belonged to space group P2₁2₁2₁, with unit-cell parameters a = 94.69, b = 102.24, c = 148.61 Å (Fig. 2 ▶).

Figure 1.

H. pylori arginase crystals grown using the hanging-drop method in 25% PEG 3350, 100 mM Bis-Tris pH 5.5, 1 mM MnCl₂, 15 mM guanidine hydrochloride. Dimensions are 0.5 × 0.1 × 0.06 mm.

Table 1. Statistics of H. pylori arginase diffraction data.

Values in parentheses are for the highest resolution shell.

Space group	P212121
Unit-cell parameters	a = 94.69, b = 102.24, c = 148.61
Wavelength (Å)	1.0000
Resolution (Å)	34.92–2.20 (2.32–2.20)
No. of reflections	73842
Rmerge†	10.1 (31.2)
Multiplicity	6.8 (6.4)
Mean I/σ(I)	11.3 (4.8)
Completeness (%)	99.9 (99.9)

^† R _merge = , where 〈I(hkl)〉 is the mean of the observations I_i(hkl) of reflection hkl.

Figure 2.

A typical diffraction pattern of an H. pylori arginase crystal.

There are probably 3–5 H. pylori arginase molecules per asymmetric unit, with corresponding Matthews coefficients of 3.33, 2.50 and 2.00 ų Da⁻¹ and solvent contents of 63.1, 50.8 and 38.5%, respectively. Self-rotation functions calculated by MOLREP in the CCP4 program suite (v.6.0.2; Winn et al., 2011 ▶) at various resolutions showed neither threefold, fourfold, fivefold nor sixfold noncrystallographic symmetry axes; however, perpendicular twofold non­crystallographic symmetry axes close to crystallographic symmetry axes were observed. Thus, we propose that there are four molecules with 222 point-group symmetry in the asymmetric unit.

The high quality of the data forms a solid foundation for our structural studies. Attempts to solve the phase problem by molecular replacement using existing models such as PDB entries 1d3v (Cox et al., 1999 ▶) and 1cev (Bewley et al., 1999 ▶) were not successful and this work is still in progress.