Purification, crystallization and preliminary X-ray crystallographic analysis of Lmo0540 from Listeria monocytogenes

The crystallization of Lmo0540 from L. monocytogenes is reported.

Abstract

Penicillin-binding proteins catalyze the biosynthesis of the peptidoglycan chains of the bacterial cell wall, which protects cells from osmotic pressure. Although Lmo0540 has been identified as a putative penicillin-binding protein that contributes to the virulence of Listeria monocytogenes, the biochemical role of Lmo0540 remains unclear. To provide insights into its biochemical function, Lmo0540 was overexpressed, purified and crystallized by the sitting-drop vapour-diffusion method. Diffraction data were collected to 1.5 Å resolution using synchrotron radiation. The crystal belonged to the C-­centred monoclinic space group C2, with unit-cell parameters a = 82.5, b = 75.7, c = 75.9 Å, α = γ = 90, β = 121.8°. A full structural determination is under way in order to elucidate the structure–function relationship of this protein.

1. Introduction

The bacterial cell wall is mainly composed of peptidoglycan chains, which play critical roles in controlling the shape of cells and in protecting them from osmotic pressure (Young, 2003 ▶; Popham & Young, 2003 ▶). These peptidoglycan chains are constantly remodelled and new chains are incorporated at specific places as the bacteria grow and divide (Macheboeuf et al., 2006 ▶). Penicillin-binding proteins (PBPs) catalyze the assembly of peptidoglycan chains through the transglycosylation and transpeptidation of lipid II, which is composed of an undecaprenyl-linked disaccharide-pentapeptide containing β-­linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) (Sauvage et al., 2008 ▶). The five-residue peptide stem is attached to the N-acetylmuramic acid. The transglycosylation reaction catalyzes the polymerization of the NAG-NAM disaccharide subunit of bacterial peptidoglycan (Halliday et al., 2006 ▶; Lovering et al., 2007 ▶), whereas the transpeptidation reaction catalyzes the cross-linking of peptidoglycan chains by a peptide bridge (Born et al., 2006 ▶). In addition, the endopeptidase activity of some PBPs is responsible for breaking peptide cross-links to allow new peptido­glycan chains to be inserted and the peptidoglycan to be recycled (Meberg et al., 2004 ▶). At present, many proteins from newly released genomic databases are annotated as putative PBPs. However, their biochemical and/or physiological roles remain unclear despite extensive studies of several representative PBPs.

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes listeriosis. It is sensitive to penicillins, but has a relatively high natural resistance to cephalosporins and fosfomycin (Conter et al., 2009 ▶; Espaze & Reynaud, 1988 ▶). Recently, nine PBPs expressed by L. monocytogenes, including Lmo0540, were shown to bind the fluorescent antibiotic BOCILLIN FL (Korsak et al., 2010 ▶). However, only a few PBPs from L. monocytogenes have been studied in order to elucidate their biochemical and/or physiological properties (Zawadzka-Skomial et al., 2006 ▶; Vicente et al., 1990 ▶; Korsak et al., 2005 ▶). Interestingly, disruption of Lmo0540 produced no defects in cell morphology but did lead to attenuated virulence, as determined from colonization of the spleen in mice (Guinane et al., 2006 ▶). Lmo0540 from L. monocytogenes was initially annotated as a low-molecular-mass (LMM) PBP homologue with variations in the classical PBP motifs. BLAST and domain analysis of Lmo0540 showed that it contained the class C β-lactamase domain. In addition, the overall fold of Lmo0540 may be most similar to the structure of the DD-peptidase (PDB entry 3pte; Kelly & Kuzin, 1995 ▶) from Streptomyces sp. R61, which shows the highest sequence identity to Lmo0540 (27%) among solved PBP structures. However, the biochemical and enzymatic properties of Lmo0540 have not been reported.

We successfully purified and crystallized the putative penicillin-binding protein Lmo0540. The resulting monoclinic crystal diffracted to 1.5 Å resolution using a synchrotron source. The crystal structure of Lmo0540 will provide insights into the biochemical function of the protein.

2. Materials and methods

2.1. Cloning, expression and purification of Lmo0540

The gene encoding the putative penicillin-binding protein Lmo0540 (UniProt accession No. Q8Y9I8) was amplified by the polymerase chain reaction (PCR) with Pfu DNA polymerase from chromosomal DNA of L. monocytogenes EGD. The primers were 5′-GAATTCCATATGGATCCTGAACCAGTAGAAGAAAAGC-3′ for the forward primer and 5′-CCCGGCTCGAGTTAATTTAAGAAACCATAAATATCATTA-3′ for the reverse primer and contained NdeI and XhoI sites, respectively (bold). The PCR product was subcloned into the pProExHTa vector (Invitrogen, USA) to generate an expression plasmid encoding residues 72–396 of Lmo0540 with an N-terminal His₆ tag. The lmo0540 gene insertion was confirmed by DNA sequencing. The resulting expression vector pProExHTa:lmo0540 was transformed into Escherichia coli BL21 (DE3) RIL cells, which were then grown at 310 K in Luria–Bertani medium containing 100 µg ml⁻¹ ampicillin and 34 µg ml⁻¹ chloramphenicol until the OD₆₀₀ reached ∼0.8. After induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside at 291 K for a further 12 h, the cells were harvested by centrifugation at 5000g at 277 K. The cell pellet was resuspended in ice-cold buffer A (30 mM Tris–HCl pH 8.0, 0.3 M NaCl) and lysed by sonication. The lysate was centrifuged at 15 000g for 30 min and the supernatant was loaded onto Ni–NTA resin (Qiagen, USA) equilibrated with buffer A. After washing with buffer A containing 20 mM imidazole, the bound protein was eluted using a solution consisting of 30 mM Tris–HCl pH 8.0, 50 mM NaCl and 200 mM imidazole. The eluted sample was digested overnight with TEV protease (Invitrogen, USA) at a ratio of 1:100(w:w) at 277 K to remove the hexahistidine tag. The target protein was bound to a 5 ml HiTrap Q column (GE Healthcare, Sweden) and was eluted with a 20-­column-volume linear gradient from 50 to 500 mM NaCl in a buffer consisting of 30 mM Tris–HCl pH 8.0 and 1 mM dithiothreitol. Finally, the protein was further purified using a Superdex 200 (GE Healthcare) column equilibrated in 10 mM Tris–HCl pH 8.0 and 100 mM NaCl. The fractions of Lmo0540 that eluted as a monomer were pooled and concentrated to 25 mg ml⁻¹. Aliquots were flash-frozen in liquid nitrogen and stored at 193 K for subsequent use in crystallization. The purity of the protein was greater than 95% based on an SDS–PAGE analysis (data not shown). The removal of the hexahistidine tag leaves three residues (GHM) encoded by the expression vector at the N-terminus of the recombinant protein.

2.2. Crystallization

Truncated Lmo0540 (325 amino acids) was crystallized using the sitting-drop vapour-diffusion method in 96-well Intelli-Plates (Hampton Research, USA). Initial crystallization conditions were obtained by sparse-matrix screening (Jancarik et al., 1991 ▶) using commercial kits from Hampton Research (California, USA) and Emerald BioSystems (Washington, USA) by manually mixing 0.4 µl protein solution and 0.4 µl reservoir solution and equilibrating against 100 µl reservoir solution at 295 K. Hits from initial crystallization screens were further optimized by varying the concentration of PEG 8000 and monobasic potassium phosphate using a VDX plate (Hampton Research, USA) to obtain crystals that were suitable for X-ray diffraction.

2.3. X-ray data collection

For X-ray data collection, a single crystal was immersed briefly into reservoir solution containing 15% glycerol as a cryoprotectant and immediately flash-cooled in a 100 K nitrogen stream. Native X-ray diffraction data were collected using an ADSC Q315r CCD detector on beamline 4A at Pohang Accelerator Laboratory (PAL; Republic of Korea) using 1° oscillations with a crystal-to-detector distance of 250 mm. The crystal was exposed for 5 s per image. A data set was collected to 1.5 Å resolution from a single crystal. The data were indexed and scaled with the HKL-2000 software package (Otwinowski & Minor, 1997 ▶). The data-collection statistics are summarized in Table 1 ▶.

Table 1. Diffraction statistics.

Values in parentheses are for the highest resolution shell. A 0σ cutoff filter was applied during the scaling process.

X-ray source	Beamline 4A, PAL
Wavelength (Å)	1.000
Resolution (Å)	30.0–1.5 (1.53–1.50)
Space group	C2
Unit-cell parameters (Å, °)	a = 82.5, b = 75.7, c = 75.9, α = γ = 90, β = 121.8
Completeness (%)	98.5 (95.1)
Rmerge†	5.0 (26.0)
Multiplicity	5.2 (3.4)
Average I/σ(I)	34.6 (3.2)

^† R _merge = , where I_i(hkl) and 〈I(hkl)〉 are the observed intensity and the mean intensity of related reflections, respectively.

3. Results and discussion

To facilitate purification and crystallization, the N-terminal 71 residues of Lmo0540, which contain the signal sequence and a putative transmembrane helix, were truncated in the expression construct. Recombinant Lmo0540 protein was expressed and purified to homogeneity using sequential chromatographic steps. The yield of purified protein was ∼10 mg per litre of E. coli culture. After optimizing the crystallization conditions, shiny single crystals were obtained using sitting-drop vapour diffusion by mixing equal volumes (2 µl) of protein solution (25 mg ml⁻¹) and reservoir solution com­posed of 23%(w/v) PEG 8000, 0.04 M monobasic potassium phosphate pH 4.5 and 3%(v/v) 1,4-dioxane. The best crystals grew within 7 d to final dimensions of approximately 0.2 × 0.2 × 0.4 mm (Fig. 1 ▶). The Lmo0540 crystal diffracted to 1.5 Å resolution and belonged to space group C2, with unit-cell parameters a = 82.5, b = 75.7, c = 75.9 Å, α = γ = 90, β = 121.8°. The diffraction data set was 98.5% complete, with an R _merge of 5.0% (Table 1 ▶). Assuming one Lmo0540 molecule per asymmetric unit, the Matthews parameter V _M (Matthews, 1968 ▶) was 2.78 ų Da⁻¹, corresponding to a solvent content of 55.8%. In order to determine the structure, molecular replacement was attempted using the structure of the DD-peptidase from Streptomyces sp. R61 (27% sequence identity for 325 aligned amino acids) as a search model in the program MOLREP (Vagin & Teplyakov, 2010 ▶), but this method was not successful. In order to solve the structure, selenomethione-substituted Lmo0540 is being prepared for phasing by multiple anomalous dispersion (MAD).

Figure 1.

Crystals of Lmo0540. The crystals grew within one week at 295 K to maximum dimensions of approximately 0.2 × 0.2 × 0.4 mm.