Cloning, purification, crystallization and preliminary X-ray analysis of ESX-1-secreted protein regulator (EspR) from Mycobacterium tuberculosis

ESX-1 secreted protein regulator (EspR, Rv3849) from M. tuberculosis has been purified and crystallized, and diffracted to 3.2 Å resolution at wavelength 0.97625 Å.

Abstract

ESX-1-secreted protein regulator (EspR; Rv3849) is a key regulator in Mycobacterium tuberculosis that delivers bacterial proteins into the host cell during infection. EspR binds directly to the Rv3616c-Rv3614c promoter and activates transcription and secretes itself from the bacterial cell by the ESX-1 system. The three-dimensional structure of EspR will aid in understanding the mechanisms by which it binds to the Rv3616c-Rv3614c promoter and is involved in transcriptional activation. This study will significantly aid in the development of EspR-based therapeutics against M. tuberculosis. The full-length EspR gene from M. tuberculosis (H37Rv strain) was cloned and overexpressed as a soluble protein in Escherichia coli. The protein was purified by affinity chromatography using His-tagged protein followed by size-exclusion chromatography. EspR was crystallized using polyethylene glycol 3350 as precipitant. The crystals diffracted to 3.2 Å resolution using synchrotron radiation of wavelength 0.97625 Å. The crystal belonged to space group P3₁21 and contained three monomers in the asymmetric unit. Native and heavy-atom-derivatized data sets were collected from EspR crystals for use in ab initio structure-solution techniques.

1. Introduction

The ESX-1 protein-secretion system of Mycobacterium tuberculosis delivers virulence factors into host macrophages and disarms them during infection (Stanley et al., 2003 ▶; Hsu et al., 2003 ▶; Pathak et al., 2007 ▶). The ESX-1 secretion system is involved in innate immune modulation after infection in macrophages (Stanley et al., 2003 ▶, 2007 ▶; MacGurn & Cox, 2007 ▶; Volkman et al., 2004 ▶). Despite the essential role of the ESX-1 system in virulence, the mechanism of ESX-1 secretion is not known. Recently, a locus containing the gene Rv3616c-Rv3614c required for ESX-1 secretion-system activity has been identified (Fortune et al., 2005 ▶; MacGurn et al., 2005 ▶). Rv3616c is a secreted substrate of the secretion-system pathway. In addition, two other substrates, ESTAT-6 and CFP-10, are also involved in the secretion pathway; however, their roles in virulence are not clear (Hsu et al., 2003 ▶; Pathak et al., 2007 ▶; de Jonge et al., 2007 ▶; Singh et al., 2003 ▶). The secretion of one substrate is dependent on the secretion of the other substrate (Fortune et al., 2005 ▶). EspR (Rv3849) is a new sub­strate of the ESX-1 system, like ESAT-6, CFP-10 and EspA, and is required for the function of the entire ESX-1 system (Fortune et al., 2005 ▶). EspR is induced upon phagocytosis and activates the expression of downstream ESX-1 components. Depletion of EspR affects M. tuberculosis gene expression, including loci that are critical for ESX-1 function (Raghavan et al., 2008 ▶).

EspR is a DNA-binding transcriptional regulator; it binds 520 bp of the Rv3616c promoter and is involved in transcriptional activation (Raghavan et al., 2008 ▶). The protein consists of 132 residues and has a molecular weight of ∼14.7 kDa. A structural homology search revealed close homology to the Bacillus subtilis transcription factor SinR, which is a helix–turn–helix DNA-binding protein (Lewis et al., 1996 ▶). The N-­terminal domain of EspR harbours a DNA-binding region and point-mutation analysis in this domain showed reduced DNA-binding affinity (Raghavan et al., 2008 ▶). The C-terminal domain of EspR is required for transcriptional activity; deletion of ten amino acids from this domain completely abolished activity (Raghavan et al., 2008 ▶).

Here, we report the cloning, purification, crystallization and preliminary X-ray crystallographic study of EspR. Structural studies of EspR will contribute significantly to understanding the mechanism of DNA binding and transcriptional activation, as well as its involve­ment in the ESX-1 secretion pathway.

2. Materials and methods

2.1. Expression and purification

The gene encoding EspR (Met1–Ala132) was amplified from M. tuberculosis (H37Rv strain) by polymerase chain reaction and cloned into pET-28a(+) vector (Novagen) containing a 6×His tag and a thrombin cleavage site at the N-terminus. The following primers were used for PCR: forward primer 5′-GATCGCTAGCATGCAA­CCGATGACCGCT-3′ and reverse primer 5′-CATGCTCGAGCTA­ATCGTCGATCCCTTC-3′. The resulting construct was transformed into Escherichia coli BL21 (DE3) cells.

The cells were grown in 2 l Luria–Bertani (LB) medium containing 50 µg ml⁻¹ kanamycin at 310 K until the OD₆₀₀ reached 0.6, followed by induction with 125 µM IPTG at 310 K for 4 h. The cultured cells were harvested by centrifugation, resuspended in 50 ml lysis buffer consisting of 25 mM Tris–HCl pH 8.0, 300 mM NaCl, 1 mM benz­amidine–HCl, 0.1% Triton X-100, 5% glycerol, 2 mM β-mercapto­ethanol, 1 mM phenylmethylsulfonyl fluoride and 0.5 mg ml⁻¹ lysozyme and disrupted by sonication at 277 K. The protein was purified using affinity chromatography and size-exclusion chromatography at 277 K (Fig. 1 ▶) and characterized by N-terminal sequencing and mass spectrometry. The purified EspR protein contained a total of 150 amino-acid residues: six residues from the 6×His tag, 12 residues from the thrombin cleavage site and 132 residues of EspR protein.

Figure 1.

(a) FPLC elution profile of the purification of EspR protein by size-exclusion chromatography using a Superdex 75 (16/60) column. The major peak corresponds to EspR protein. (b) SDS–PAGE analysis after size-exclusion chromatography of purified EspR protein. Lane M, molecular-weight markers (kDa). Lanes 1 and 2, SDS–PAGE analysis of the eluted fractions containing purified EspR protein.

2.2. Crystallization

The EspR protein was concentrated to 8 mg ml⁻¹ in 20 mM Tris–HCl pH 7.5 for crystallization experiments. The initial crystallization conditions were screened using Structure Screens I and II from Molecular Dimensions and Crystal Screen, Crystal Screen 2 and PEG/Ion Screen from Hampton Research. All crystallization experiments were performed at 277 K using the sitting-drop vapour-diffusion technique. In each trial, 1 µl EspR protein solution was mixed with 1 µl precipitant solution and equilibrated against a reservoir containing 100 µl precipitant solution.

2.3. Data collection

For intensity data collection, a single crystal of EspR was transferred into a solution consisting of 30% PEG 3350, 200 mM sodium malonate and 100 mM bis-tris propane pH 6. These crystals were directly frozen in liquid nitrogen as 30% PEG 3350 was suitable as a cryoprotectant for diffraction measurements at cryogenic temperature.

A native intensity data set was collected from an EspR crystal at 100 K using a MAR225 image-plate detector on the BM14 beamline at the ESRF, France. Indexing and integration of the images were performed using the DENZO program and scaling and merging were performed using the SCALEPACK program (Otwinowski & Minor, 1997 ▶). F _obs values were produced using the SCALEPACK2MTZ program from the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶).

3. Results and discussion

EspR crystals were obtained from a drop comprising 1 µl protein solution and 1 µl reservoir solution consisting of 20% PEG 3350, 200 mM sodium malonate and 100 mM bis-tris propane pH 6.5. The EspR crystals usually appeared after 7–8 d and grew to maximum dimensions of 0.4 × 0.3 × 0.2 mm (Fig. 2 ▶). The crystals often grew with a rectangular shape. In a few drops the crystals grew as clusters and a unique fragment was separated by touching the cryoloop on the surface of the crystals.

Figure 2.

Trigonal crystals of EspR protein. The crystals grew as rectangular bars with typical dimensions of 0.4 × 0.3 × 0.2 mm.

The native crystal belonged to space group P3₁21, with unit-cell parameters a = b = 83.9, c = 131.0 Å, α = β = 90, γ = 120°, and contained three monomers in the asymmetric unit. Diffaction data were collected to 3.2 Å resolution (Fig. 3 ▶) and details of the data-collection and processing statistics are given in Table 1 ▶. Based on the presence of three molecules of EspR in the asymmetric unit, the Matthews coefficient was V _M = 2.6 ų Da⁻¹, which corresponds to a solvent content of 53.3%. These values lie within the range normally observed in protein crystals (Matthews, 1968 ▶).

Figure 3.

Typical X-ray diffraction pattern of a native EspR crystal (oscillation width 1°). The edge of the frame corresponds to 2.7 Å resolution.

Table 1. X-ray data-collection statistics.

Values in parentheses are for the last resolution shell.

Resolution (Å)	50–3.2 (3.26–3.20)
X-ray source	BM14, ESRF
Wavelength (Å)	0.97625
Space group	P3121
Unit-cell parameters (Å, °)	a = b = 83.9, c = 131.0, α = β = 90, γ = 120
Observed reflections	84807
Unique reflections	8730 (309)
Completeness (%)	97.3 (76.3)
Multiplicity (%)	9.7
Rmerge (%)†	13.3 (66.9)
Average I/σ(I)	14.1 (1.8)

^† R _merge = , where I_i(hkl) is the ith intensity measurement of reflection hkl and 〈I(hkl)〉 is the average intensity of that reflection.

To obtain phase information, molecular-replacement analysis was performed using the Phaser program (McCoy et al., 2005 ▶). Two models, (i) residues 613–693 of enterochelin esterase from Shigella flexneri (PDB entry 2b20, chain A; Y. Kim, N. Maltseva, I. Dementieva, P. Quartey, D. Holzle, F. Collart & A. Joachimiak, unpublished work) with 32% sequence identity and (ii) B. subtilis transcription factor SinR (PDB entry 1b0n; Lewis et al., 1998 ▶) with 19.7% sequence identity, were obtained using the MODWEB homology-modelling web server (http://salilab.org/modeller). Molecular-replacement analysis with both models did not yield useful phases for structure solution of EspR.

Currently, we are collecting heavy-atom derivative data sets, including a selenomethionine derivative, of EspR and expect to solve the structure by ab initio methods. The three-dimensional structure of EspR will aid in understanding its structure–function relationship, which will play a key role in therapeutics against M. tuberculosis.