Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

Abstract

<jats:title>Abstract</jats:title> <jats:p>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.</jats:p>

Non‐Carbohydrate Glycomimetics as Inhibitors of Calcium(II)‐Binding Lectins

“…Protein‐observed 19 F (PrOF) NMR spectroscopy is a sensitive technique to spot weak binders in a low millimolar affinity range. Here, we used previously established PrOF NMR with 5‐fluorotryptophan‐labeled (5FW)‐LecA [26] to determine the binding region of catechols. First, we measured 5FW‐LecA in presence of para ‐nitrophenyl β‐ d ‐galactoside (pNPGal) as well‐known ligand for the carbohydrate‐binding site of LecA.…”

19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

“… 101 However, this is not always the case, where even weak ligands can exhibit slow chemical exchange binding. 102 In chemical shift perturbation experiments, nonspecific effects such as protein aggregation or denaturation can be readily detected which are indicated by a global decrease in resonance intensity or coalescence of protein resonances which helps to identify potential false positives in screens. 41 …”

“…Such a case was observed for the carbohydrate binding protein LecA studied by Shanina et al for lectin inhibitor development. 102 LecA was metabolically labelled with 5FW at all four native tryptophan residues, of which W42 and W33 are near the binding site of the natural carbohydrate ligand, d -galactose. 5FW-LecA was titrated with known weak binder, N -acetyl d -galactosamine.…”

Druggable Allosteric Sites in β‐Propeller Lectins

“…[15] Previously,t his method proved to be valuable for identification of small molecules targeting ab acterial lectin LecA. [25] Given that BambL monomer contains six tryptophan residues in the carbohydrate-binding site (Figure S10 a), we sought to apply PrOF NMR to verify the impact of fragment binding on the orthosteric site.F or this,w e substituted tryptophan residues in BambL with 5-fluorotryptophanes (5FW) and assigned four out of six 5FW by sitedirected mutagenesis (Figure S10 b). Next, we confirmed the activity of 5FW BambL using MeFuc and 2FF,w here all six 5FWs showed as low exchange on the NMR time scale (Figure S10 c,d).…”