Role of Water Molecules in Structure and Energetics of Pseudomonas aeruginosa Lectin I Interacting with Disaccharides

Anne Imberty,Bertrand Blanchard,Stefan Oscarson,Alessandra Nurisso,Aymeric Audfray,Annabelle Varrot,Lina Rydner

doi:10.1074/jbc.m110.108340

Abstract

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an alpha-galactose residue at their nonreducing end, such as the disaccharides alphaGal1-2betaGalOMe, alphaGal1-3betaGalOMe, and alphaGal1-4betaGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL.alphaGal1-2betaGalOMe complex, which was solved at 2.4 A resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1-2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.

Highlights

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is the major mortality factor for cystic fibrosis patients, causing endobronchial infections and related neutrophilic inflammatory responses [3]
We previously described the structural basis and thermodynamic properties of this lectin in complex with oligosaccharide moieties of glycosphingolipids combining several approaches such as cell surface labeling, glycan array analysis, titration microcalorimetry, crystallography, and molecular modeling [11]
The crystal structure of PA-IL in complex with ␣Gal1–2␤GalOMe was solved at 2.4 Å resolution, and thermodynamic of PA-IL binding to the three oligosaccharides was measured by titration microcalorimetry

Summary

Introduction

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is the major mortality factor for cystic fibrosis patients, causing endobronchial infections and related neutrophilic inflammatory responses [3]. The bacterium produces two soluble lectins, lectin I and lectin II (PA-IL and PA-IIL), both located in the cytoplasm and on the outer membrane [4]. These lectins are expressed under the control of the quorum sensing system and are considered as virulence factors that have been proposed to be involved in adhesion to glycoconjugates on respiratory epithelia and biofilm formation [5, 6]. We previously described the structural basis and thermodynamic properties of this lectin in complex with oligosaccharide moieties of glycosphingolipids combining several approaches such as cell surface labeling, glycan array analysis, titration microcalorimetry, crystallography, and molecular modeling [11]. The modeling data, in agreement with the experimental one, showed the importance of one structural bridge water molecule always present in the PA-IL binding site

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