Crystallization and preliminary X-ray diffraction studies of the transcriptional repressor PaaX, the main regulator of the phenylacetic acid degradation pathway in Escherichia coli W

PaaX, the main regulator of the phenylacetic acid degradation pathway in bacteria, has been crystallized using the sitting-drop vapour-diffusion method. Preliminary X-ray data to 2.3 Å resolution allowed determination of its unit-cell parameters and space group and initiation of its structure determination.

Abstract

PaaX is the main regulator of the phenylacetic acid aerobic degradation pathway in bacteria and acts as a transcriptional repressor in the absence of its inducer phenylacetyl-coenzyme A. The natural presence and the recent accumulation of a variety of highly toxic aromatic compounds owing to human pollution has created considerable interest in the study of degradation pathways in bacteria, the most important microorganisms capable of recycling these compounds, in order to design and apply novel bioremediation strategies. PaaX from Escherichia coli W was cloned, overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 0.9 M Li₂SO₄ and 0.5 M sodium citrate pH 5.8. These crystals, which belonged to the monoclinic space group C2 with unit-cell parameters a = 167.88, b = 106.23, c = 85.87 Å, β = 108.33°, allowed the collection of an X-ray data set to 2.3 Å resolution.

1. Introduction

After carbohydrates, aromatic compounds are the most abundant class of organic compounds used by nature (Harwood & Parales, 1996 ▶) and their concentrations have recently increased in many ecosystems as a consequence of human pollution (OECD, 1994 ▶). Microorganisms, mostly bacteria, recycle both natural and man-made aromatic compounds. In this sense, the degradation pathways used by these microorganisms are highly relevant in the field of bio­remediation, which uses microorganisms or their enzymes to eliminate pollutants from contaminated environments (Ramos et al., 1994 ▶; Harwood & Parales, 1996 ▶; Pieper & Reineke, 2000 ▶).

In bacteria, aerobic degradation of phenylacetic acid (PAA) begins with the esterification of PAA with coenzyme A (CoA) by phenyl­acetyl-CoA (PA-CoA) ligase, which is common to anaerobic degradation pathways (Luengo et al., 2001 ▶), in contrast to the degradation of hydroxylated PAA derivatives. In Escherichia coli W, paa gene clusters code for enzymes that aerobically degrade PAA (Ferrández et al., 1998 ▶). Homologues of paa genes have been found in different microorganisms, all of which have similar regulation mechanisms (García et al., 2000 ▶; Rost et al., 2002 ▶; Navarro-Llorens et al., 2005 ▶; del Peso-Santos et al., 2006 ▶; Di Gennaro et al., 2007 ▶).

The transcriptional repressor PaaX (315 amino acids, 35 295 Da; Ferrández et al., 1998 ▶) encoded in one of the paa clusters represses the expression of the PAA aerobic degradation pathway enzymes, binding to the operator sequences in the paa gene clusters in the absence of PA-CoA and therefore blocking transcription by RNA polymerase. When the PA-CoA concentration increases the repressor is released from the DNA, allowing transcription to proceed. Additionally, PaaX regulates the expression of degradation pathways that produce PAA as their final product, such as penicillin G acylase (PGA), which hydrolyses numerous esters and amines of phenyl­acetic acid (Galán et al., 2004 ▶; Kim et al., 2004 ▶), and the styrene-degradation pathway in Pseudomonas sp. strain Y2 (del Peso-Santos et al., 2006 ▶). Hence, PaaX is currently considered to be a central regulator in the aerobic degradation of aromatic compounds derived from PAA in several microorganisms (Luengo et al., 2001 ▶).

PaaX is classified in the Pfam database as a member of the PaaX-like protein family (http://pfam.sanger.ac.uk/family/PF07848), which encompasses transcriptional regulators of the PAA-degradation pathway in several proteobacteria. The N-terminal region of PaaX-like proteins has sequence homology to the winged helix–turn–helix (wHTH) motif of the GntR family of transcriptional regulators, while the C-terminal region bears no homology to other protein families (Ferrández et al., 2000 ▶; Tropel & van der Meer, 2004 ▶). Therefore, it has been speculated that PaaX comprises two domains, with the N-­terminal domain being in charge of binding DNA and repressing transcription, while the C-terminal domain binds the PA-CoA activator and regulates the activity of the N-terminal domain (Ferrández et al., 2000 ▶). Interestingly, the proposed palindromic consensus DNA-binding sequence of PaaX has a separation of 25 bp between the two halves of the palindrome (Kim et al., 2004 ▶), which is unprecedented in transcriptional regulators of the GntR family.

Although the function of PaaX has been thoroughly studied, structural studies are absent from the literature. Only two structures from the PaaX-like protein family have been deposited in the PDB (PDB entries 3l09 from Jannaschia sp. and 3kfw from Mycobacterium tuberculosis; Joint Center for Structural Genomics, unpublished work; U. A. Ramagopal, R. Toro, S. K. Burley & S. C. Almo, un­published work) and preliminary crystallization and X-ray analyses have been reported for a PaaX-like protein (Cao et al., 2009 ▶). However, no further analysis of these structures has been published.

In this work, we report the crystallization and preliminary X-ray diffraction studies of a cysteine-lacking variant of PaaX from E. coli W.

2. Experimental

2.1. E. coli PaaX expression and purification

A hexahistidine-tagged cysteine-lacking variant of PaaX, HisPaaX C4, was purified from pX2BSc0 plasmid in E. coli MV1184 cells (Sambrook & Russell, 2001 ▶). This mutant conserves its DNA-binding and transcription-activation functionality, as well as the overall structure and stability of the wild-type form (Hernández-Rocamora et al., unpublished results). A fresh transformant colony was grown overnight at 310 K. The culture was diluted 100-fold in 1 l LB medium and grown at 303 K until the optical density at 600 nm reached 0.5–0.6. Overexpression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a concentration of 1 mM followed by further incubation at 303 K for 16 h.

Cultures were harvested by centrifugation at 3000g for 15 min at 277 K. The cells were resuspended in 40 ml Im20 buffer (50 mM sodium phosphate pH 8 with 300 mM NaCl and 20 mM imidazole) and sonicated on ice in a Branson 250 sonicator. The soluble protein fraction was separated by centrifugation at 100 000g for 1 h at 277 K.

Supernatants were mixed in batches with 5 ml chelating Sepharose (Chelating Sepharose Fast Flow, GE Healthcare) loaded with Ni and gently stirred for 1 h at room temperature (RT). The Sepharose with the absorbed protein was packed into a column and washed consecutively with two column volumes of Im20 buffer and two column volumes of Im100 buffer (as Im20 but with 40 mM imidazole). PaaX was eluted with Im400 buffer (20 mM Tris–HCl pH 8 with 500 mM NaCl and 400 mM imidazole). The fractions containing the highest concentrations of PaaX (measured using UV absorbance; the purity was checked by SDS–PAGE) were pooled. Samples were frozen quickly in a dry-ice/ethanol bath and stored at 193 K.

2.2. Crystallization

Initial crystallization assays based on the commercial screens JCSG+ Suite and PACT Suite from Qiagen and Crystal Screen and Crystal Screen 2 from Hampton Research were carried out by high-throughput techniques using a NanoDrop ExtY robot (Innovadyne Technologies Inc.). All crystallization trials were performed by the sitting-drop vapour-diffusion method at 291 K in Innovaplate SD-2 microplates (Innovadyne Technologies Inc.), mixing equal amounts (250 nl) of PaaX solution at 9.7 mg ml⁻¹ [20 mM Tris pH 8, 500 mM NaCl, 400 mM imidazole and 20%(v/v) glycerol] and precipitant solution. The drops (500 nl) were equilibrated against 65 µl precipitant solution. Optimized crystals were obtained by scaling up the drop volume using the sitting-drop method: 1 µl PaaX solution was mixed with 1 µl precipitant solution and equilibrated against 500 µl precipitant solution.

2.3. Diffraction data collection and processing

Crystals were soaked for 5 s in a cryoprotectant solution [20%(v/v) glycerol in the crystallization mother liquor] prior to flash-cooling at 100 K by direct transfer into liquid nitrogen using a cryogenic system. X-ray data sets were collected using synchrotron radiation at the ESRF, Grenoble, France. A data set was collected from a PaaX crystal to 2.3 Å resolution on beamline ID14-1 (λ = 0.93340 Å) using an ADSC Quantum Q210 detector (oscillation range 1°, crystal-to-detector distance 191.64 mm). The diffraction data were processed using the XDS program (Kabsch, 2010 ▶) and scaled using SCALA (Evans, 1993 ▶) from the CCP4 program suite (Winn et al., 2011 ▶); processing data are summarized in Table 1 ▶.

Table 1. Data-collection statistics for PaaX crystals.

Values in parentheses are for the highest resolution shell.

Crystal data
Space group	C2
Unit-cell parameters
a (Å)	167.88
b (Å)	106.23
c (Å)	85.87
β (°)	108.33
Data processing
Temperature (K)	100
Wavelength (Å)	0.9334
Resolution (Å)	44.2–2.3 (2.4–2.3)
Unique data	63547
Multiplicity	3.7 (3.7)
Data completeness (%)	99.9 (99.9)
Average I/σ(I)	20.9 (2.8)
Molecules in asymmetric unit	4
Matthews coefficient (Å3 Da−1)	2.89
Solvent content (%)	61
Rmerge†	0.05 (0.49)

^† R _merge = , where I_i(hkl) is the ith measurement of reflection hkl and 〈I(hkl)〉 is the weighted mean of all measurements.

3. Results

A fully functional PaaX variant containing an N-terminal hexahistidine tag and with all four cysteine residues present in the wild-type protein substituted by alanines was constructed (Hernández-Rocamora et al., unpublished results) in order to increase the stability during purification and to avoid aggregation at the concentrations required for crystallization purposes.

PaaX microcrystals grew from condition No. 15 of Crystal Screen 2 (1.0 M lithium sulfate, 0.5 M ammonium sulfate and 0.5 M sodium citrate pH 5.6). Good-quality crystals of PaaX were obtained from optimized conditions (0.9 M lithium sulfate and 0.5 M sodium citrate pH 5.8; Fig. 1 ▶). An X-ray data set was collected to 2.3 Å resolution. The images displayed good-quality diffraction patterns (Fig. 2 ▶). Preliminary X-ray data processing showed good statistics (Table 1 ▶). The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 167.88, b = 106.23, c = 85.87 Å, β = 108.33°. Specific volume calculations based on the molecular weight of PaaX (32 kDa) and the unit-cell parameters (volume = 1 458 706.8 ų) indicated the possible presence of four monomer molecules in the asymmetric unit with 61% solvent content (V _M = 2.89 ų Da⁻¹; Matthews, 1968 ▶).

Figure 1.

PaaX crystals obtained with 0.9 M lithium sulfate and 0.5 M sodium citrate pH 5.8 under polarized light. The approximate dimensions of the crystals are 0.15 × 0.4 × 0.4 mm.

Figure 2.

(a) X-ray diffraction pattern of PaaX crystals; (b) an enlargement showing high-resolution spots.

Structural determination of PaaX by the molecular-replacement method using the structural model from Jannaschia sp. (PDB entry 3l09; Joint Center for Structural Genomics, unpublished work), which shows 30% sequence homology, is currently in progress.