Background: Lectins 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.

The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.

In this study, in silico mutagenesis and docking in Ralstonia solanacearum lectin (RSL) were carried out, and the ability of several docking software programs to calculate binding affinity was evaluated. In silico mutation of six amino acid residues (Agr17, Glu28, Gly39, Ala40, Trp76, and Trp81) was done, and a total of 114 in silico mutants of RSL were docked with Me-α-L-fucoside. Our results show that polar residues Arg17 and Glu28, as well as nonpolar amino acids Trp76 and Trp81, are crucial for binding. Gly39 may also influence ligand binding because any mutations at this position lead to a change in the binding pocket shape. The Ala40 residue was found to be the most interesting residue for mutagenesis and can affect the selectivity and/or affinity. In general, the docking software used performs better for high affinity binders and fails to place the binding affinities in the correct order.

Summary: The new version of the TRITON program provides user-friendly graphical tools for modeling protein mutants using the external program MODELLER and for docking ligands into the mutants using the external program AutoDock. TRITON can now be used to design ligand-binding proteins, to study protein–ligand binding mechanisms or simply to dock any ligand to a protein.Availability: Executable files of TRITON are available free of charge for academic users at http://ncbr.chemi.muni.cz/triton/Contact: triton@chemi.muni.czSupplementary information: Supplementary data are available at Bioinformatics online.

This article is focused on the application of two types of docking software, AutoDock and DOCK. It is aimed at studying the interactions of a calcium-dependent bacterial lectin PA-IIL (from Pseudomonas aeruginosa) and its in silico mutants with saccharide ligands. The effect of different partial charges assigned to the calcium ions was tested and evaluated in terms of the best agreement with the crystal structure. The results of DOCK were further optimized by molecular dynamics and rescored using AMBER. For both software, the agreement of the docked structures and the provided binding energies were evaluated in terms of prediction accuracy. This was carried out by comparing the computed results to the crystal structures and experimentally determined binding energies, respectively. The performance of both docking software applied on a studied problem was evaluated as well. The molecular docking methods proved efficient in identifying the correct binding modes in terms of geometry and partially also in predicting the preference changes caused by mutation. Obtaining a reasonable in silico method for the prediction of lectin-saccharide interactions may be possible in the future.

Computers in chemistry V 0380In silico Mutagenesis and Docking Studies of Pseudomonas aeruginosa PA-IIL Lectin -Predicting Binding Modes and Energies. -(ADAM, J.; KRIZ, Z.; PROKOP, M.; WIMMEROVA, M.; KOCA*, J.; J. Chem. Inf. Model. (J. Chem. Inf.

This article focuses on designing mutations of the PA-IIL lectin from Pseudomonas aeruginosa that lead to change in specificity. Following the previous results revealing the importance of the amino acid triad 22-23-24 (so-called specificity-binding loop), saturation in silico mutagenesis was performed, with the intent of finding mutations that increase the lectin's affinity and modify its specificity. For that purpose, a combination of docking, molecular dynamics and binding free energy calculation was used. The combination of methods revealed mutations that changed the performance of the wild-type lectin and its mutants to their preferred partners. The mutation at position 22 resulted in 85% in inactivation of the binding site, and the mutation at 23 did not have strong effects thanks to the side chain being pointed away from the binding site. Molecular dynamics simulations followed by binding free energy calculation were performed on mutants with promising results from docking, and also at those where the amino acid at position 24 was replaced for bulkier or longer polar chain. The key mutants were also prepared in vitro and their binding properties determined by isothermal titration calorimetry. Combination of the used methods proved to be able to predict changes in the lectin performance and helped in explaining the data observed experimentally.

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