Computational protocol: Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex

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[…] Protein was mixed with DNA in a 1:1:1.2 molar ratio (ParC55:ParE30:18-mer E-site DNA) with an overall concentration of 4 mg ml−1. Levofloxacin and magnesium chloride were added to final concentrations of 2 and 10 mM, respectively. The mixture was pre-incubated at room temperature overnight. Initial crystallization screening was performed by sitting-drop vapour diffusion in a 96-well MRC crystallization plate (600 nl protein mixture + 400 nl reservoir solution) using a Mosquito robot (TTP Labtech; http://www.ttplabtech.com). The best crystals were obtained using capillary counter-diffusion against 50 mM sodium cacodylate pH 6.5, 2.5% Tacsimate (Hampton Research; McPherson & Cudney, 2006), 7% 2-propanol, 62.5 mM KCl, 7.5 mM MgCl2 at 304 K. The crystals were flash-cooled at 100 K in cryoprotectant buffer C [50 mM sodium cacodyl­ate pH 6.5, 2.5% Tacsimate, 62.5 mM KCl, 7.5 mM MgCl2, 1 mM β-mercapto­ethanol, 30%(v/v) MPD]. The best data set was collected on beamline I03 at Diamond Light Source at a wavelength of 0.9763 Å using an ADSC Quantum 315 detector. The data extended to 2.6 Å resolution anisotropically and were used in refinement with a maximum-likelihood target in the initial refinement cycles; they were deposited in the PDB without introducing a resolution cutoff. However, owing to the high R merge values in the outer shells, the final resolution is given as 2.9 Å and the statistics are reported according to this ‘trimmed’ resolution. The resolution cutoff was based on the rejection criteria R merge < 50% and I/σ(I) > 1.5 in the highest resolution shell. The data were integrated using HKL-2000 (Otwinowski & Minor, 1997). The space group was determined to be P3121, with unit-cell parameters a = b = 157.83, c = 211.15 Å.The structure was solved by molecular replacement using Phaser (McCoy et al., 2007) as implemented within the CCP4 suite (Winn et al., 2011) and our previously published topo­isomerase IV–levofloxacin structure (PDB entry 3k9f; Laponogov et al., 2010). Refinement was performed in PHENIX (Adams et al., 2002, 2010) with manual inspection and corrections performed in WinCoot (Emsley & Cowtan, 2004; Emsley et al., 2010).The structure was verified using WinCoot and PROCHECK (Laskowski et al., 1993). [...] ParC55/ParE30 protein stock in incubation buffer (at 4.5 mg ml−1) was mixed with the ‘pre-cut’ 34-mer DNA stock in a 1:1.2 protein:DNA molar ratio. High-concentration stocks of levofloxacin and MgCl2 were added to give final concentrations of 2 and 10 mM, respectively. The mixture was incubated overnight at room temperature. Initial crystallization screening was performed by sitting-drop vapour diffusion in 96-well MRC crystallization plates (600 nl protein mixture + 300 nl reservoir solution) using a Mosquito robot. When the optimal crystallization conditions had been established, conventional hanging-drop vapour diffusion in 24-well Linbro plates (4 µl protein mixture + 2 µl reservoir solution) was used to increase the crystal size.Crystals formed after ∼7–10 d at room temperature. The crystallization conditions varied slightly from batch to batch in the range 0.1 M Tris pH 7.5–8.0, 0–50 mM NaCl, 4–8% PEG 4000, 12–15% glycerol.It should be mentioned that several other DNA oligomers with the same binding-site sequence were tried for crystallization (i.e. 20-mer, ‘pre-cut’ 20-mer and 34-mer DNA sequences). However, these protein–DNA–drug complexes did not produce good-quality crystals for data collection.Crystals were tested in-house for diffraction quality using an Oxford Xcalibur Nova CCD diffractometer and were then transported for high-resolution data collection at Diamond Light Source (Harwell Science and Innovation Campus, Oxfordshire, England). The data were collected on beamline I03 (wavelength 0.9762 Å) using a Pilatus 6M-F detector (0.2° oscillation per image, 100 K nitrogen stream). The best crystals diffracted to ∼3.2 Å resolution.All data sets were integrated with MOSFLM (Leslie & Powell, 2007) and merged with SCALA (Evans, 2006) as implemented in CCP4 (Winn et al., 2011). The ParC55/ParE30–DNA–levofloxacin crystals belonged to space group P21, with unit-cell parameters a = 102.07, b = 161.53, c = 138.60 Å, α = 90.00, β = 94.22, γ = 90.00°. They contained two ParC/ParE–DNA heterodimers in the asymmetric unit.Several data sets were collected, some of which contained visible diffraction to 3.2 Å resolution, but owing to potential internal twinning and space-group ambiguity (most data sets could be integrated in space groups P21 and P212121) and the fact that the structure solution could be obtained in both space groups, careful selection of the integration ranges as well as appropriate data truncation were necessary. The best region of data was integrated to 3.35 Å (see Table 3 for statistics). The resolution cutoff was based on the rejection criteria R merge < 50% and I/σ(I) > 1.5 in the highest resolution shell.The structure was solved by the molecular-replacement method in Phaser (McCoy et al., 2007) using the levofloxacin–DNA cleavage complex of topoisomerase IV from S. pneumoniae as a search model (PDB entry 3rae; ∼41.8% sequence identity). Refinement was performed in PHENIX (Adams et al., 2002, 2010) using secondary-structure restraints derived by superposition of the K. pneumoniae ParC/ParE model with the previously solved complex of S. pneumoniae ParC/ParE. Rigid-body, positional and TLS refinements were performed. Levofloxacin molecules and magnesium ions were placed during the final stages of refinement based on missing electron density in the σA-weighted 2F obs − F calc and F obs − F calc maps. WinCoot (Emsley & Cowtan, 2004) was used for interactive model fitting. The structure was verified using WinCoot and PROCHECK (Laskowski et al., 1993). The resulting model had good geometry, with 87.8, 9.9 and 1.3% of residues in the favoured, allowed and generously allowed regions of the Ramachandran plot, respectively, and no more than 1% of residues in disallowed regions. The data-collection and final refinement statistics are given in Tables 3 and 4. Sequence alignment was performed in ClustalW (Larkin et al., 2007, McWilliam et al., 2013). Figures were prepared using PyMOL (DeLano, 2008), CHEMDRAW (Evans, 2014) and CorelDRAW (http://www.coreldraw.com). […]

Pipeline specifications

Software tools HKL-2000, PROCHECK, Clustal W, PyMOL, ChemDraw, CorelDraw
Applications Drug design, Miscellaneous, Protein structure analysis
Organisms Klebsiella pneumoniae, Streptococcus pneumoniae, Bacteria
Diseases Infection, Klebsiella Infections, Lung Diseases, Meningitis, Pneumonia
Chemicals Fluoroquinolones, Levofloxacin