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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 December 1; 64(Pt 12): 1153–1155.
Published online 2008 November 28. doi:  10.1107/S1744309108036555
PMCID: PMC2593690

Cloning, purification, crystallization and preliminary X-ray analysis of a bacterial GABA receptor with a Venus flytrap fold

Abstract

In response to infection by the pathogen Agrobacterium tumefaciens, plants synthesize several stress amino acids, including γ-aminobutyric acid (GABA), which modulates the expression of bacterial virulence factors. GABA penetrates into the bacterial cytoplasm via an ABC transporter that is associated with the periplasmic receptor Atu2422. Mature receptor Atu2422 (without its signal peptide) was overexpressed in Escherichia coli, purified and crystallized. A complete data set was collected to 1.35 Å resolution at 100 K. The crystals belonged to the monoclinic space group C2 and contained one molecule in the asymmetric unit. Molecular replacement was performed and the initial electron-density maps revealed a closed form of this Venus flytrap (VFT) receptor, suggesting the presence of an endogenous E. coli ligand.

Keywords: Atu2422, GABA receptors, Venus flytrap fold, Agrobacterium tumefaciens

1. Introduction

The pathogen Agrobacterium tumefaciens is responsible for crown gall disease in numerous plants of agronomical interest (reviewed by Escobar & Dandekar, 2003 [triangle]). Several plant signals are required to control the expression of virulence. γ-Aminobutyric acid (GABA) acts as a plant signal that attenuates virulence functions by inducing the expression of the lactonase AttM in A. tumefaciens (Chevrot et al., 2006 [triangle]; Yuan et al., 2008 [triangle]). This lactonase can cleave the bacterial signal 3-oxooctanoyl homoserine lactone (OC8HSL) when A. tumefaciens colonizes the plant tissues. Therefore, it may affect the ex­pression of OC8HSL-dependent functions, such as the dissemination of virulence genes by horizontal transfer of the tumour-inducing (Ti) plasmid (Zhang et al., 2002 [triangle]) and the number of emerging tumours at the infection site (Chevrot et al., 2006 [triangle]).

The import of GABA into several bacteria requires a periplasmic bacterial receptor that traps the GABA molecule and transfers it to the ABC transporter Bra. The GABA receptor is called Atu2422 in A. tumefaciens strain C58 (Chevrot et al., 2006 [triangle]) and BraC (Rl3745) in Rhizobium leguminosarum bv. viciae strain 3841 (Hosie et al., 2002 [triangle]). The peptide sequence of Atu2422 shares around 40% sequence identity with two Escherichia coli periplasmic amino-acid receptors of known structure: Leu-Ile-Val-binding protein (LIVBP, LivJ; Sack, Saper et al., 1989 [triangle]; Trakhanov et al., 2005 [triangle]) and leucine-binding protein (LBP, LivK; Sack, Trakhanov et al., 1989 [triangle]; Magnusson et al., 2004 [triangle]). These amino-acid receptors, which consist of two domains separated by a deep cleft, exhibit the Venus flytrap (VFT) architecture, which is conserved among a large number of ligand-binding proteins in bacteria and eukaryotes, including the GABAB receptors in mammals (Galvez et al., 1999 [triangle]), as shown by modelling based on sequence alignment (Acher & Bertrand, 2005 [triangle]). The unliganded receptor adopts an open form, whereas it adopts a closed form upon ligand binding.

Here, we report the successful purification, crystallization and preliminary X-ray analysis of the bacterial GABA receptor Atu2422.

2. Cloning, expression and purification

Genomic DNA purified from A. tumefaciens strain C58 was used as a template to amplify the DNA encoding the mature protein Atu2422 (consisting of 349 residues without its signal peptide). Suitable restriction sites (NdeI and NotI; shown in bold) were added to the primers (5′-GGAATTCCATATGGATGTCGTCATCGCTGTC and 5′-TTTGCGGCCGCTTAATGGTGATGGTGATGGTGGCTGCCCTGCTGG) for cloning into a pET-9aSN1 expression vector (a gift from S. Chéruel, IBBMC, University Paris Sud, Orsay, France). An oligonucleotide coding for a six-histidine tag was added to the 3′ end of the gene to facilitate protein purification.

The PCR product was purified with a QIAquick PCR purification kit (Qiagen), digested with NdeI and NotI enzymes and cloned into the expression vector pET-9aSN1, which was digested with the same enzymes. The construct was then introduced by heat shock into XL1-Blue chemically competent cells. The nucleotide sequence of one selected clone was confirmed by DNA-sequence analysis.

For the expression of Atu2422 (residues 24–372)-6×His, transformed BL21 (DE3) STAR E. coli cells were grown at 310 K to an OD600 of about 0.8 in 2×TY medium containing 50 µg ml−1 kanamycin. Expression of the His-tagged receptor was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside. The cells were grown for a further 3 h at 310 K before centrifugation (4000g, 10 min). The pellet was resuspended in 25 mM Tris–HCl pH 8.5, 150 mM NaCl. Cell lysis was achieved by sonication after the addition of an antiprotease cocktail (Sigma). Cell debris was spun out by centrifugation at 40 000g for 30 min at 277 K. Recombinant mature protein with a C-­terminal His tag (Atu2422-His) was purified by IMAC chromatography using Ni–NTA resin (Qiagen) equilibrated in 25 mM Tris–HCl pH 7.5, 300 mM NaCl, 10 mM imidazole. Low-affinity binding con­taminants were washed from the column with 35 mM imidazole prior to the elution of Atu2422 with 300 mM imidazole. The eluted sample was further purified by size-exclusion chromatography using a Superdex 200 HiLoad column (Amersham Biosciences) equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl. Both chromatographic steps were performed on an ÄKTA Purifier 10 (Amersham Biosciences) at 277 K. The purity of the sample was determined by SDS–PAGE. Atu2422-His was concentrated to 11 mg ml−1 using Vivaspin-10 centrifugal concentrators (Vivasciences) and stored frozen.

3. Crystallization

In crystallization trials, mature Atu2422-His protein was used at a concentration of 11 mg ml−1 in a buffer consisting of 20 mM HEPES pH 7.5 and 150 mM NaCl. Commercial crystallization kits (the PEG II and Classics Suites from Qiagen) were screened in sitting-drop vapour-diffusion experiments using a nanodrop robot (Cartesian Proteomic Solutions) at 293 K. Small crystals appeared within 3 d in only one condition (PEG II condition D4). Crystallization conditions were then manually optimized with home-made solutions. 2 µl protein solution was mixed with 2 µl reservoir solution consisting of 0.1 M Tris–HCl pH 8.5, 35%(w/v) PEG 4000, 100 mM CaCl2 and equilibrated over 1 ml reservoir solution, leading to large thin plate-shaped crystals (Fig. 1 [triangle]).

Figure 1
Large thin plate-shaped crystals of Atu2422 (dimensions 0.6 × 0.1 × 0.04 mm).

4. Data collection and processing

Crystals were flash-frozen in a cryoprotectant solution containing 0.1 M Tris–HCl pH 8.5, 37% PEG 4000, 100 mM CaCl2 and 20% glycerol. Data-collection experiments were carried out at 100 K on the PROXIMA I beamline at SOLEIL (Saint Aubin, France). 360° of data were collected in 1° frames, with 1 s exposure per frame. Diffraction intensities were evaluated with the program XDS (Kabsch, 1993 [triangle]) and further processed using the CCP4 program suite (Collaborative Computational Project, Number 4, 1994 [triangle]). The crystal diffracted to 1.35 Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 116.2, b = 38.9, c = 71.6 Å, β = 95.7°. Data-collection and processing statistics are given in Table 1 [triangle].

Table 1
Data-collection statistics

5. Molecular replacement

The asymmetric unit can contain only one molecule, corresponding to a Matthews coefficient (Matthews, 1968 [triangle]) of 2.15 Å3 Da−1 and a solvent content of 42.9%. Molecular replacement using the program Phaser (Storoni et al., 2004 [triangle]) gave an incorrect solution (RFZ = 5.2, TFZ = 2.8, LLG = −70 and an initial R factor of 51%) using the apo structure of E. coli LBP (PDB code 1usg) as a search model. Taking into account the fact that periplasmic amino-acid receptors are known to bind endogenous amino acids during the production/purification procedure (Trakhanov et al., 2005 [triangle]; Huvent et al., 2006 [triangle]), a liganded form of LBP (PDB code 1usk; leucine-bound) was used as a model and led to a solution with good scores (RFZ = 9.5, TFZ = 10.2, LLG = 158) and an initial R factor of 44%. Examination of the resulting model and density maps clearly showed that Atu2422 adopts a closed conformation, meaning that the receptor was crystallized with a bound fortuitous ligand that needs to be identified. Refinement of the mature receptor Atu2422 structure and determination of its amino-acid selectivity are under way.

Acknowledgments

We are grateful to Andrew Thompson for help in data collection on PROXIMA I. We thank Véronique Henriot for the expression test, which was realised on the LEBS cloning/protein-expression facility. The crystallization work benefitted from the LEBS facilities of the IMAGIF Structural Biology and Proteomic Unit at the Gif campus (http://www.imagif.cnrs.fr).

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Articles from Acta Crystallographica Section F: Structural Biology and Crystallization Communications are provided here courtesy of International Union of Crystallography