Abstract
The lipid A biosynthesis pathway is essential in Pseudomonas aeruginosa. LpxA and LpxD are the first and third enzymes in this pathway respectively, and are regarded as promising antibiotic targets. The unique structural similarities between these two enzymes make them suitable targets for dual-binding inhibitors, a characteristic that would decrease the likelihood of mutational resistance and increase cell-based activity. We report the discovery of multiple small molecule ligands that bind to P. aeruginosa LpxA and LpxD, including dual-binding ligands. Binding poses were determined for select compounds by X-ray crystallography. The new structures reveal a previously uncharacterized magnesium ion residing at the core of the LpxD trimer. In addition, ligand binding in the LpxD active site resulted in conformational changes in the distal C-terminal helix-bundle, which forms extensive contacts with acyl carrier protein (ACP) during catalysis. These ligand-dependent conformational changes suggest a potential allosteric influence of reaction intermediates on ACP binding, and vice versa. Taken together, the novel small molecule ligands and their crystal structures provide new chemical scaffolds for ligand discovery targeting lipid A biosynthesis, while revealing structural features of interest for future investigation of LpxD function.
Highlights
Antibiotic resistance is a worldwide threat that challenges our ability to successfully treat bacterial infection, exhausting health care resources and worsening patient prognosis
The concept of dual targeting the early steps of the lipid A biosynthetic pathway has previously been demonstrated with a peptide molecule RJPXD33, found to inhibit both LpxA and LpxD when expressed in E. coli[27]
We report the discovery of several small molecules that bind to both P. aeruginosa LpxA and LpxD with μM affinity, identified using a targeted structure-based methodology that utilizes molecular docking, surface plasmon resonance (SPR) bioanalysis, and high-resolution X-ray crystallography
Summary
Antibiotic resistance is a worldwide threat that challenges our ability to successfully treat bacterial infection, exhausting health care resources and worsening patient prognosis. While LpxC is not homologous to LpxA and LpxD, the latter two enzymes share several unique structural features, consistent with their functional similarities in catalyzing the transfer of a 10 or 12 carbon chain fatty acid from ACP to UDP-GlcNAc through a concerted acid-base mechanism[16,17]. Both proteins form biological homotrimers that contain a left-handed helical fold comprised of multiple parallel β-sheets[14,16,17,18,19,20,21,22,23,24]. The structural analysis has provided valuable insights regarding inhibitor binding hot spots of LpxA and LpxD, and allosteric effects induced by ligand binding in the LpxD active site
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