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Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.
Shanina, Elena Siebs, Eike Zhang, Hengxi Silva, Daniel Varón Joachim, Ines Titz, Alexander Rademacher, Christoph
Shanina, Elena
Siebs, Eike
Zhang, Hengxi
Silva, Daniel Varón
Joachim, Ines
Titz, Alexander
Rademacher, Christoph
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2020-06-23
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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.
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Glycobiology. 2020;cwaa057. doi:10.1093/glycob/cwaa057.
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en
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1460-2423
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Attribution-NonCommercial-ShareAlike 4.0 International