Structures ofPseudomonas aeruginosaβ-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

Abstract

Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinantPseudomonas aeruginosaβ-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

An Experimental Toolbox for Structure-Based Hit Discovery for P. Aeruginosa FabF, a Promising Target for Antibiotics

“…Further, we have successfully optimized our previous crystallization conditions for PaFabF to allow for soaking of small molecules in DMSO containing solutions. [36] We routinely obtain crystals diffracting to 1.7 Å allowing for detailed binding information for ligands. Nevertheless, for only one of the virtual screening hits a complex structure could be obtained.…”

“…For this purpose, protein constructs containing an AviTag [37] that can be biotinylated using the biotin ligase BirA [38] were designed and purified as described earlier. [36] Avi-tagged PaFabF C164Q was prone to precipitation and thus not accessible for BLI experiments The remaining biotinylated PaFabF variants were immobilized on Super Streptavidin (SSA) biosensors. Binding of ligands at different concentrations was recorded as wavelength shift of the interference pattern of white light reflected from a layer of immobilized protein on the biosensor tip, and an internal reference layer.…”

“…Starting from our previously established crystallization conditions for PaFabF, [36] protein and precipitant concentration as well as the drop-to-buffer ratio were optimized to allow for reliable crystallization and soaking of ligands. As a proof of concept, PaFabF C164Q was soaked with platensimycin and the structure of the complex was determined at a resolution of 1.78 Å (Table S1).…”

“…The protein constructs for FabF containing an AviTag [37] for BLI experiments (here called PaAviFabF) were designed as follows: N-terminal His6-tag, TEV cleavage site, Avi-tag, followed by linker residues (GS). All FabF variants were expressed as described earlier, [36] with only minor deviations. The plasmids were heat-shock transformed (45 s, 315 K) into Rosetta (DE3) pLysS competent E. coli cells (Novagen) and cultured in autoinduction medium (1L LB with 50 mL 20X NPS, 20 mL 50X 5052 and 1 mL MgSO4) supplemented with chloramphenicol (1 mL 30 mg/mL) and kanamycin (1 mL 50 mg/mL) for 48 h at 293 K with shaking at 250 rpm.…”

“…The target organism was chosen as there is a particular high need for new antibiotics to fight infections caused by carbapenem-resistant P. aeruginosa. [2] Starting from our previous structural experience with PaFabF [36] we have established robust crystallization conditions that allow for soaking with ligands. Further, we have developed a binding assay using bio-layer interferometry (BLI).…”