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 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.
Highlights
CR LecA forms a protein homotetramer (Fig. 1A) and requires a calcium (II) ion (Ca2+) to S coordinate binding to its natural monosaccharide ligand, D−galactose (D−Gal) (Cioci and U others 2003)
NC Here, we explored protein−observed 19F (PrOF) NMR using LecA labeled with 5FW (5FW LecA) to detect Ubinding of ligands with moderate as well as low affinities
Our affinity data in fluorescence polarization (FP) assay for ligands, in RE particular D−Gal, was in a close range 1230 200 μM and 1991 μM for both unlabeled R LecA and 5FW LecA, respectively (Table SIV). This result suggests that O the affinities for D−GalNAc derived from the FP assay for LecA and 5FW LecA diverged C from PrOF NMR due to higher sensitivity of 19F NMR to spot weak binders and UNshows the advantages of PrOF NMR in discovery of weak interactions
Summary
Our affinity data in FP assay for ligands, in RE particular D−Gal, was in a close range 1230 200 μM and 1991 μM for both unlabeled R LecA and 5FW LecA, respectively (Table SIV) This result suggests that O the affinities for D−GalNAc derived from the FP assay for LecA and 5FW LecA diverged C from PrOF NMR due to higher sensitivity of 19F NMR to spot weak binders and UNshows the advantages of PrOF NMR in discovery of weak interactions. PrOF NMR has proven R more sensitive for identification of weak ligands like D−GalNAc due to chance to observe O the formation of a protein−ligand complex in NMR at earlier time point compared to FP C assay These results represent the first studies demonstrating the potential UNof 5FW LecA PrOF NMR to assess binding of weak ligands.
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