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

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

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

Glycobiology. 2020 Jun 23;:

Authors: Shanina E, Siebs E, Zhang H, Silva DV, Joachim I, Titz A, Rademacher C

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


PMID: 32573695 [PubMed - as supplied by publisher]



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