Recombinant production, crystallization and X-ray crystallographic structure determination of peptidyl-tRNA hydrolase from Salmonella typhimurium

Abstract

Peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) from the pathogenic bacterium Salmonella typhimurium has been cloned, expressed in Escherichia coli and crystallized for X-ray analysis. Crystals were grown using hanging-drop vapor diffusion against a reservoir solution consisting of 0.03 M citric acid, 0.05 M bis-tris propane, 1% glycerol, 3% sucrose, 25% PEG 6000 pH 7.6. Crystals were used to obtain the three-dimensional structure of the native protein at 1.6 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P2₁2₁2₁ with unit-cell parameters a=62.1, b=64.9, c=110.5 Å, α=β=γ=90°. The asymmetric unit of the crystallographic lattice was composed of two copies of the enzyme molecule with a 51% solvent fraction, corresponding to a Matthews coefficient of 2.02 Å3 Da(-1). The structural coordinates reported serve as a foundation for computational and structure-guided efforts towards novel small-molecule Pth1 inhibitors and potential antibacterial development.

Keywords: Salmonella typhimurium; peptidyl-tRNA; peptidyl-tRNA hydrolase 1.

Figure 1

Pth1 purification and functional assay. (a) 12% SDS–PAGE gel. Lane MW, molecular-weight marker with sizes of the control proteins in kDa labeled. Lanes 1–6, Ni²⁺-affinity chromatography purification: lane 1, unbound fraction; lane 2, 25 mM imidazole wash; lanes 3–6, eluted fractions containing S. typhimurium Pth1; lane 7, total lysate; lane 8, soluble fraction of the lysate; lane 9, insoluble portion of the lysate; lane 10, S. typhimurium Pth1 after size-exclusion chromatography. (b) Functional assay for S. typhimurium Pth1 activity: bulk peptidyl-tRNA (lane 1) was cleaved by the purified recombinant enzyme (lane 2). The control band of 5S ribosomal rRNA in the samples is indicated.

Figure 2

Crystal of S. typhimurium Pth1 used for X-ray diffraction. The crystal dimensions were approximately 0.15 × 0.07 × 0.8 mm. Crystals were obtained within 3 d of 18°C incubation.

Figure 3

Structural comparison. (a), (b) The high-resolution structure of S. typhimurium Pth1 (PDB entry 4p7b). (c), (d) Overlay of the high-resolution S. typhimurium Pth1 structure (blue) with structures of other bacterial Pth1s (gray): E. coli Pth1 (PDB entry 2pth; Schmitt et al., 1997 ▶), P. aeruginosa Pth1 (PDB entry 4fyj; Hughes et al., 2012 ▶), F. tularensis Pth1 (PDB entry 3nea; Clarke et al., 2011 ▶), M. smegmatis Pth1 (PDB entry 3p2j, Kumar et al., 2012 ▶) and Acinetobacter baumannii Pth1 (PDB entry 3wh4, Kaushik et al., 2013 ▶). The regions with high variations in tertiary structure between the enzymes are indicated with arrows. (e), (f) Surface representation of S. typhimurium Pth1 with residues colored depending on their conservation in bacterial Pth1 enzymes for which high-resolution structures have been solved: red, strictly conserved amino-acid residues; orange, highly homologous residues; yellow, homologous residues; gray, nonhomologous residues. The orientation of the molecule in the surface representation corresponds to the ribbon diagrams.

Figure 4

S. typhimurium Pth1 B factors. (a) S. typhimurium Pth1 structure with amino-acid residues labeled based on their B factors. The B-factor color scale is shown in (a). Regions discussed in the text are shown magnified: (b) helix 4 with flanking loops and (c) region of amino-acid residues 48–63.