Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding

Abstract

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

Discovery of N-β-l-Fucosyl Amides as High-Affinity Ligands for the Pseudomonas aeruginosa Lectin LecB

“…In general, α-linked substituents at the anomeric center of fucose point toward the solvent when bound to LecB, while β-linked substituents are oriented toward the protein surface, and thus, the observed difference in binding entropy possibly resulted from displaced protein-bound water molecules for β-benzamide 4 . Favorable entropy of binding is very unusual in protein–carbohydrate interactions but appears to be a signature of this class of two-calcium lectins as rationalized by analysis of water dynamics through neutron crystallography . Optimizing this favorable thermodynamic contribution through water displacement appears here as a valuable strategy.…”

“…Favorable entropy of binding is very unusual in protein–carbohydrate interactions but appears to be a signature of this class of two-calcium lectins 54 as rationalized by analysis of water dynamics through neutron crystallography. 55 Optimizing this favorable thermodynamic contribution through water displacement appears here as a valuable strategy. The low flexibility of the β-glycosyl amide containing ligand is also a favorable factor to avoid an entropy barrier.…”

Multivalent glycocyclopeptides: conjugation methods and biological applications

“…LecA and LecB are tetrameric proteins containing a total of four binding sites for Gal and Fuc, respectively, and thus represent key targets to develop antiadhesive agents as an alternative to antibiotic treatments. [99][100][101] Other fucose-binding proteins have been identified in pathogens such as Burkholderia ambifaria and Aspergillus fumigatus. Among them, B. ambifaria BambL 102 and A. fumigatus AFL (FleA) 103 have the peculiarity of including the repetition of similar b-sheets.…”

Structural Considerations for Building Synthetic Glycoconjugates as Inhibitors for Pseudomonas aeruginosa Lectins

“…This particularly high affinity comes at the cost of a loss in specificity; this lectin also recognises d ‐mannose and d ‐arabinose, among others [24] . The Fuc O2, O3 and O4 atoms coordinate Ca 2+ ions in the binding pocket and furthermore O5, O1, O2 and O3 form an intricate H‐bonding network with neighbouring residues, particularly Asp99, Asp96, Asp104, Ser23 and structural water (Figure 1d) [8,25] . The shortest distance between neighbouring Fuc‐binding sites is ∼40 Å.…”