Structural Basis of the Preferential Binding for Globo-Series Glycosphingolipids Displayed by Pseudomonas aeruginosa Lectin I

Abstract

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for α-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward αGal1–4βGal-terminating structures and weaker binding to αGal1–3βGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P k. Trisaccharide moieties of Gb3 (αGal1–4βGal1–4Glc) and isoglobotriaosylceramide (αGal1–3βGal1–4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to αGal1–3βGal1–4Glc trisaccharide has been solved at 1.9-Ǻ resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward αGal1–3Gal with that toward αGal1–4Gal. Docking studies demonstrated that αGal1–4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.