Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

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.