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 it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (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 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 ?M). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl ?-d-galactoside (pNPGal, Kd = 54 ± 6 ?M) demonstrated a 6-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.

SUBMITTER: Shanina E 

PROVIDER: S-EPMC7874386 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature