Crystal structure of Pseudomonas aeruginosa FabB C161A, a template for structure-based design for new antibiotics

Protein expression and purification

The gene coding for P. aeruginosa PA14 FabB C161A was synthesised and cloned in a bacterial plasmid pET-28a(+)-TEV vector after a DNA sequence coding for a 6-His-tag followed by a TEV cleavage site. To find good expression conditions, seven widely used E. coli strains were transformed with the plasmid (BL21 (DE3), BL21 (DE3) pLysS, C41 (DE3), C41 (DE3) pLysS, C43 (DE3), C43 (DE3) pLysS and Rosetta (DE3) pLysS) and screened for protein expression. The best results were obtained with Rosetta (DE3) pLysS cells (data not shown). Therefore, this cell line was used for all subsequent protein expression experiments.²¹

His-tagged PaFabB C161A was purified using affinity chromatography with a Ni column followed by size exclusion chromatography (SEC). To obtain FabB with a cleaved His-tag, the protein obtained after affinity chromatography was cleaved with TEV protease. The cleaved protein was separated from the protease and the tag by inverse affinity chromatography followed by SEC. In both cases, pure protein was obtained as judged by SDS-PAGE gel electrophoresis (Figure 2). Typical yields for His-tagged PaFabB C161A were 26 mg/L and for cleaved PaFabB C161A 7 mg/L.

Figure 2. Protein samples on an SDS−PAGE gel.

Lane 1: PaFabB C164A (without His-tag) after inverse affinity chromatography, lane 2: 6-His-tagged PaFabB C164A after SEC purification, lane 3: PaFabB C164A (without His-tag) after SEC purification, lane 4: protein ladder.

Crystallization of PaFabB C161A

Crystallization trials of His-tagged PaFabB C161A and cleaved PaFabB C164A were attempted using the JCSG+, PACT premier, HELIX (only His-tagged PaFabB C161A) and LFS screens. No promising crystallization conditions for His-tagged PaFabB C161A were found using these screens. In contrast, 11 different conditions resulted in crystals of His-tag cleaved PaFabB C164A (Table 1, Figure 2). All of these conditions contained PEG 3350 between 20 and 25% and a number of conditions contained ethylene glycol. Further, the majority of the conditions contained 0.1 M Bis-Tris propane, and 0.2 M sodium iodide. Therefore, these components were kept for further optimization trials. The pH of the initial conditions varied from 5.5 to 8.5. As crystals grown in a buffer of pH 7.5 were visually judged to be more regular (e. g. the crystal shown in Figure 3B), this pH was fixed during optimization. These considerations resulted in an optimization matrix where the concentration of PEG 3350 was varied between 5 and 30% and the protein concentration between 9 and 23 mg/mL. Ethylene glycol was added to all conditions at either 10 or 20% while 0.2 M sodium iodide and 0.1 M Bis-Tris propane were fixed (Figure 4). Under 32 conditions, crystals were obtained. These were mounted and used for diffraction experiments.

Figure 3. Selected crystals obtained from various screens.

A) condition F3 from LFS, B) G3 from LFS, C) F3 from PACT premier, D) G3 from PACT premier (for composition of crystallization buffer see Table 1).

Figure 4. Plate layout for optimization of crystallization conditions.

The numbers in the cells indicate the ratio between protein solution and crystallization buffer in the drops (drop 1-1:1 ratio, drop 2-1:2 ratio, drop 3-2:1 ratio). Coloured cells indicate conditions from which crystal were harvested and mounted for diffraction experiments. Green cells indicate conditions under which diffracting crystals were obtained.

Six different conditions led to well-diffracting crystals (Figure 4). For these, data sets with resolutions between 2 and 1.3 Å could be collected. For the best diffracting crystal, the crystal structure was determined using a homology model created based on Vibrio cholerae FabB (VcFabB PDB Id 4XOX) as search model (Table 2). The crystal was in the space group C 2 2 21 and contained 2 protein molecules in the asymmetric unit.

Crystal structure of PaFabB C161A

PaFabB C161A has the same overall fold as observed before for FabB and FabF from other organisms (Figure 5 and Figure 6A). The rmsd between PaFabB C161A and VcFabB (the protein with the highest sequence identity in the PDB (72%), PDB Id 4XOX) is 0.424 Å while the rmsd to the w.t. PaFabF (sequence identity 41%, PDB Id 4JPF) is 0.843 Å. The two catalytic histidines, His 296 and His 331, are highly conserved and well aligned with the catalytic histidines from both VcFabB as well as the ones from PaFabF (Figure 6B).

Figure 5. Cartoon representation showing the tertiary structure of PaFabB C161A.

Beta sheets and alpha helices are shown in yellow and red colour respectively while loops are shown in green.

Figure 6. Alignment of VcFabB (PDB Id 4XOX), PaFabF (PDB Id 4JPF) and PaFabB C161A.

A) The three different enzymes are shown in orange, green and magenta cartoon style, respectively. B) Alignment of the active site catalytic triad of VcFabB, w. t. PaFabF and PaFabB C161A.

Due to the high concentration of ethylene glycol (20% v/v) and salt in the well and protein buffers, respectively (150 mM NaCl and 200 mM NaI), 18 ethylene glycol molecules and 11 ions (Cl^- and I^-) were identified and placed in the crystal structure of PaFabB C161A during refinement (Figure 7A). Some of these molecules were found to bind in the active site of the protein (Figure 7B). The chloride ion Cl 1 binds tightly (B factor for Cl 1 is 18 Ų, average B-factor for protein atoms is 16.8 Ų, average ions B factors is 30.7 Ų) in the active site of chain B, in close proximity to the catalytic residues His 296 (3.3 Å) and His 331 (3. 2Å). Moreover, Cl 1 forms two additional interactions with an ethylene glycol (EDO511, average B factor 18 Ų) and a water molecule (HOH227) in the active site.

Figure 7. Crystal structure of PaFabB C161A.

A) The structure of PaFabB is shown as magenta cartoon style. Iodine and chloride ions are shown with deep purple and green colour respectively. B) Active site residues are shown as magenta sticks, water molecules and chloride ions are shown as red and green spheres, respectively, while the distances between the chloride ion Cl 1 and the neighbouring molecules are shown as yellow dashed lines.

Active site and differences to PaFabF

Phe 400/391 (numbering based on PaFabF/PaFabB) was previously identified, as one of the highly conserved active site residues, to play a pivotal role in substrate specificity and ligand binding, by adopting a different conformation between the w.t. and the intermediate state of the FabF enzyme.^9,12 In w. t. PaFabF (PDB Id 4JPF, Figure 8A and B), Phe400 is in a ‘closed’ conformation (dihedral angle C-CA-CB-CG = -177.1^o). When mutating the catalytic residue Cys 164 to Gln 164 (PDB Id 7OC1 – Figure 8A and C) the enzyme has been shown to mimic the intermediate state and to trap the Phe 391 into the ‘open’ conformation (dihedral angle C-CA-CB-CG = 168.8^o). Here, the catalytic residue Cys 161 of PaFabB was mutated to Ala 161. As can be seen from the crystal structure (Figure 8A and D), Phe 391 adopts the ‘open’ conformation as expected for an intermediate-mimicking FabB variant (dihedral angle C-CA-CB-CG = 170.2^o). Although, the overall sequence identity between PaFabB and PaFabF is only 41%, the conservation in the active site is much higher (Figure 9). Apart from Thr 271 in FabF that is mutated to Val 268 in FabB, all active site residues involved in hydrogen-bond interactions with platensimycin are conserved between the two enzymes (Figure 8A). That makes it highly likely that ligands binding into the active site of FabF may also bind to FabB with a similar affinity, and thus opens up the possibility for the designing of dual inhibitors for both FabF and FabB that will lead to a complete inhibition of the last step of the fatty acid elongation cycle.

Figure 8. Alignment of PaFabF (PDB Id 4JPF), PaFabF C164Q (PDB Id 7OC1) and PaFabB C161A.

A) The active site residues of the three different enzymes are shown as green, cyan and magenta sticks, respectively. Platensimycin binding to PaFabF C164Q (PDB Id 7OC1) is shown as yellow sticks. Side chain conformation and dihedral angle C-CA-CB-CG of Phe391/400 is shown in B) for PaFabF C) for PaFabF C164Q and D) for PaFabB C161A.

Figure 9. Sequence identity conservation between PaFabB C161A and PaFabF (PDB Id 4JPF).

A) Alignment between chain A of PaFabF and PaFabB C161A is shown as cartoon style and color-coded based on the sequence identity between the two isoenzymes. B) Zoom in showing the active site and active site residues. Residue labels and numbering are based on PaFabB only.

Recombinant protein production and purification

The gene coding for P. aeruginosa PA14 FabB (ORF number (open reading frame): PA14_43690), with a single point mutation C161A was synthesised and cloned in a bacterial plasmid pET-28a(+)-TEV vector using the cloning sites NdeI/BamHI by Genscript. The plasmid had a DNA sequence coding for a 6-His-tag followed by a TEV cleavage site before PaFabB. Seven different E. coli strains (OverExpress C41(DE3) SOLOs and C43(DE3) SOLOs from Biosearch technologies; BL-21(DE3), BL-21(DE3) pLysS, C41(DE3) pLysS and C43(DE3) pLysS from Lucigen, and Rosetta (DE3) pLysS from Merck) were heat-shock transformed with the synthesised plasmid. Expression of PaFabB in each transformed cell line was tested as per manufacturer protocol.

E.coli Rosetta (DE3) pLysS competent cells yielded the highest protein expression, based on SDS-PAGE analysis, and were used as an expression system for large-scale protein production and purification. Transformed cells were inoculated in 50 mL of LB medium supplemented with kanamycin (30 μg/mL) and chloramphenicol (50 μg/mL) overnight at 310 K. Pre-culture stocks were prepared by mixing the overnight culture with glycerol (final concentration 40% v/v), aliquoted and kept in –80 °C until use. For large-scale expression, 0.1 mL of pre-culture stock was inoculated in 100 mL of LB medium supplemented with kanamycin (30 μg/mL) and chloramphenicol (50 μg/mL) overnight at 310 K. The entire volume was then transferred into 900 mL of LB-medium containing antibiotics and the cell growth continued until OD₆₀₀ reached 0.7. Protein expression was then induced by adding IPTG to a final concentration of 1 mM and the expression continued for another 3-3.5 hours.

Cells were harvested by centrifugation (15 minutes, 5000 g, 277 K), resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, 10% glycerol (v/v), pH 7.4) with addition of one tablet of Complete EDTA-free protease inhibitor cocktail (Roche) and incubated with magnet stirring for 60 minutes at 277 K. 20 U (units) of DNAse I (Sigma Aldrich) was added per cell pellet, before the mixture was sonicated on ice by an ultrasonic processor (Sonics, Vibra-Cell VC130) for a total of two minutes with 10 seconds pulses with amplitude 70. The debris and insoluble protein were pelleted by centrifugation at 15000 rpm, 277 K, for 30 minutes. The supernatant was collected and filtered with Whatman filter units 0.2 μM (GE healthcare) using a syringe. The protein was then purified using a Ni²⁺ Sepharose High Performance HisTrap HP 5 mL column (GE Healthcare) with an increasing imidazole gradient from 0 to 500 mM. The fractions containing PaFabB C161A were pooled and TEV protease was added to remove the affinity tag. The mixture was dialyzed with buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl) overnight at 277 K and the cleaved protein was purified by passage through a Ni²⁺ HisTrap column. SEC was then performed on a HiLoad 26/600 Superdex 75 pg column (Cytiva) with equilibration buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4). Purity was confirmed by SDS–PAGE (Mini-PROTEAN TGX Stain-Free Precast Gel; Bio-Rad) and the final concentration of PaFabB C161A was determined using a NanoDrop ND-1000 (Thermo Fisher Scientific).

Crystallization and X-ray data collection

For crystallization trials JCSG+ (MD1-37), PACT premier (MD1-29) and LFS (Ligand Friendly Screen, MD1-122) crystallization screens from Molecular Dimensions were used. His-tag cleaved PaFabB C161A (23 mg/mL) in 20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4, was mixed with well buffer in different ratios (2:1, 1:1 and 1:2) on a Triple Sitting Drop 96-well plate (TTP Labtech) using a crystallography Mosquito LCP (TTP LabTech). The plates were incubated at 20°C. Optimization (Figure 4) of the initial hit conditions (Table 2) was achieved by varying the precipitants and protein concentrations while keeping the salt and buffer concentration constant. Optimisation led to rod-shaped crystals (250 × 100 × 10 μm) in multiple drops (Figure 3).

Crystals with a final concentration of precipitant lower than 25%(w/v) were cryoprotected with a mixture consisting of the crystallization buffer and Cryomix 9 from CryoSol MD1-90 (Molecular Dimensions) (final composition of the cryomixture: 0.2 M NaI, 0.1 M Bis-Tris propane pH 7.5, 5%(w/v) PEG 3350, 10% (v/v) EG 5 % (v/v), diethylene glycol, 5 % (v/v) 1,2-propanediol, 5 % (v/v) dimethyl sulfoxide, 5 % (v/v) glycerol, 5 mM NDSB 201 (3-(1-Pyridinio)-1-propanesulfonate), 5 % (v/v) 1,4-dioxane) prior to flash-cooling in liquid nitrogen.

X-ray data were collected from single crystals at the DESY synchrotron (Hamburg, Germany) at the P11 high-throughput MX beamline. In each case, crystals were maintained at 100 K and the X-ray wavelength was 0.976246 Å. Data were processed with the automatic data processing pipeline of P11 beamline, using XDS.¹³

Structure solution and refinement

The structure was solved by molecular replacement using Dimple¹⁴ from the CCP4i2 suite.¹⁵ As search model, a homology model generated from wt. VcFabB (PDB Id 4XOX) with 72% sequence identity was used. Refinement was performed using REFMAC5¹⁶ while inspection of electron-density and difference density maps and model manipulation was achieved using Coot.¹⁷ During refinement, water molecules, ions and side-chain conformers were included. The model geometry was assessed using MolProbity,¹⁸ the PDB redo server¹⁹ and the PDB validation tools. The crystallographic data and refinement statistics are listed in Table 2. The figures were generated with PyMOL v.2.4.1 (Schrödinger, LLC) and VMD v.1.9.3.²⁰