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Abstract

The carbohydrate−binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA−mediated biofilms is desired, but limited to carbohydrate−based ligands. Moreover, discovery of drug−like ligands for LecA is challenging due to its weak affinities. Therefore, we established a protein−observed 19F (PrOF) NMR to probe ligand binding to LecA. LecA was labeled with 5 − fluoroindole to incorporate 5 − fluorotryptophanes and the resonances were assigned by site−directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca²⁺ and d − galactose. Following NMR resonance perturbation of W42, which is located in the carbohydrate−binding region of LecA, allowed to monitor binding of low affinity ligands such as N − acetyl d − galactosamine (d − GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a six-fold improved binding of d − Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.