Martina Pokorná

Structural Basis for the Interaction between Human Milk Oligosaccharides and the Bacterial Lectin Pa-Iil of Pseudomonas Aeruginosa.

One of the mechanisms contributing to the protection by breast-feeding of the newborn against enteric diseases is related to the ability of human milk oligosaccharides to prevent the attachment of pathogenic bacteria to the duodenual epithelium. Indeed, a variety of fucosylated oligosaccharides, specific to human milk, form part of the innate immune system. In the present study, we demonstrate the specific blocking of PA-IIL, a fucose-binding lectin of the human pathogen Pseudomonas aeruginosa, by milk oligosaccharides. Two fucosylated epitopes, Lewis a and 3-fucosyl-lactose (Lewis x glucose analogue) bind to the lectin with dissociation constants of 2.2x10(-7) M and 3.6x10(-7) M respectively. Thermodynamic studies indicate that these interactions are dominated by enthalpy. The entropy contribution is slightly favourable when binding to fucose and to the highest-affinity ligand, Lewis a. The high-resolution X-ray structures of two complexes of PA-IIL with milk oligosaccharides allow the precise determination of the conformation of a trisaccharide and a pentasaccharide. The different types of interaction between the oligosaccharides and the protein involve not only hydrogen bonding, but also calcium- and water-bridged contacts, allowing a rationalization of the thermodynamic data. This study provides important structural information about compounds that could be of general application in new therapeutic strategies against bacterial infections.

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

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal 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 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A 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 alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal 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.

High affinity fucose binding of Pseudomonas aeruginosa lectin PA‐IIL: 1.0 Å resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches

PA‐IIL is a fucose‐binding lectin from Pseudomonas aeruginosa that is closely related to the virulence factors of the bacterium. Previous structural studies have revealed a new carbohydrate‐binding mode with direct involvement of two calcium ions (Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Peréz S, Wu AM, Gilboa‐Garber N, Imberty A. Structural basis for selective recognition of oligosaccharides from cystic fibrosis patients by the lectin PA‐IIL of Pseudomonas aeruginosa. Nat Struct Biol 2002;9:918–921). A combination of thermodynamic, structural, and computational methods has been used to study the basis of the high affinity for the monosaccharide ligand. A titration microcalorimetry study indicated that the high affinity is enthalpy driven. The crystal structure of the tetrameric PA‐IIL in complex with fucose and calcium was refined to 1.0 Å resolution and, in combination with modeling, allowed a proposal to be made for the hydrogen‐bond network in the binding site. Calculations of partial charges using ab initio computational chemistry methods indicated that extensive delocalization of charges between the calcium ions, the side chains of the protein‐binding site and the carbohydrate ligand is responsible for the high enthalpy of binding and therefore for the unusually high affinity observed for this unique mode of carbohydrate recognition. Proteins 2005.

Binding of different monosaccharides by lectin PA-IIL from Pseudomonas aeruginosa : Thermodynamics data correlated with X-ray structures

The lectin from Pseudomonas aeruginosa (PA‐IIL) is involved in host recognition and biofilm formation. Lectin not only displays an unusually high affinity for fucose but also binds to l‐fucose, l‐galactose and d‐arabinose that differ only by the group at position 5 of the sugar ring. Isothermal calorimetry experiments provided precise determination of affinity for the three methyl‐glycosides and revealed a large enthalpy contribution. The crystal structures of the complexes of PA‐IIL with l‐galactose and Met‐β‐d‐arabinoside have been determined and compared with the PA‐IIL/fucose complex described previously. A combination of the structures and thermodynamics provided clues for the role of the hydrophobic group in affinity.

Engineering of PA-IIL lectin from Pseudomonas aeruginosa – Unravelling the role of the specificity loop for sugar preference

BackgroundLectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose – a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action.ResultsIn vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22–23–24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions – both of afore mentioned have strong effects on the saccharide preferences.ConclusionMutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.

Importance of oligomerisation on Pseudomonas aeruginosaLectin-II binding affinity. In silico and in vitro mutagenesis

The effect of terminal GLY114* deletion on the binding affinity of the PA-IIL lectin toward l-fucose was investigated. Both experimental (isothermal titration calorimetry) and computational (molecular dynamics simulations) methods have shown that the deletion mutation decreases the L-fucose affinity. It implies that the PA-IIL saccharide binding affinity is influenced by the dimerization of the lectin. A detailed analysis of computational data confirms the key role of electrostatic interactions in the PA-IIL/saccharide binding.