Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

• Published in: Glycobiology (Oxford) 2021-02, Vol.31 (2), p.159-165
• Main Authors: Shanina, Elena, Siebs, Eike, Zhang, Hengxi, Varón Silva, Daniel, Joachim, Ines, Titz, Alexander, Rademacher, Christoph
• Format: Article
• Language: English
• Subjects: Adhesins, Bacterial - analysis; Carbohydrate Conformation; Fluorine-19 Magnetic Resonance Imaging; Models, Molecular; Pseudomonas aeruginosa - chemistry; Recombinant Proteins - analysis; Structural Biology
• ISSN: 1460-2423; 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwaa057

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 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) 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.