1 Structural and thermodynamic basis of Pseudomonas aeruginosa lectin II (PAII-L) interacting with different monosaccharides Charles Sabin1, Michaela Wimmerová2, Martina Budová2, Lenka Snajdrová2, Catherine Gautier1, Jaroslav Koča2, Edward Mitchell3, Nechama Gilboa-Garber4 & Anne Imberty1 1CERMAV – CNRS, BP 53, 38041Grenoble Cedex 9, France 2NCBR, Masaryk University, Kotlarska 2, Brno, Czech Republic 3ESRF, Experiment Division, BP 220,F Grenoble Cedex, France 4Bar-Ilan University, Faculty of Life Sciences, Ramat Gan 52900,Israel Introduction PA IIL lectin Pseudomonas aeruginosa produces two soluble lectins, PAI-L (LecA) and PAII-L (LecB) which recognise galactose and fucose respectively [2]. These lectins are associated with the internal membrane of P. aeruginosa, but a fraction is also exposed at the surface of the bacteria. The production of these lectins is associated with those of cytotoxic virulence factors [3]. The gene lecA coding for the lectin was identified and its sequence completely characterised [4]. Inhibition of haemagglu-tination studies associated with equilibrium dialysis revealed that the affinity for fucose is much larger, than that generally observed for lectins interacting with monovalent ligands. Crystallogenesis and X-ray diffraction PAII-L/Met--D-Arabinoside The chronic colonisation of the lungs by Pseudomonas aeruginosa is a major cause of infections and of mortality for cystic fibrosis (CF) patients. The genetic change responsible for the disease modifies, by an unknown mechanism, the processes of glycosylation. In the CF patients lungs, the modifications appear in glycoconjuguates of epithelial cell surface as well as in pulmonary and salivary mucins. The diffraction data collections were carried out at ESRF, Grenoble on beamlines ID14-1 and ID14-2, at 100K. Phasing of PA-IIL/fuc structure was performed by the single-wavelength anomalous diffraction (SAD) method after soaking the crystal in holmium. After initial phasing by holmium SAD, high resolution (1.0Å) data was phased using the calcium ion positions derived from the holmium and low resolution native data sets in ACORN to determine the initial phases. All other PA-IIL/monosaccharides structures were solved by molecular replacement with the use of the AMORE software. Diffraction image Pseudomonas aeruginosa Tetramer PAII-L/fucose The overall structure is a tetramer composed of 4 chains of 114 amino acids. Each monomer is complexed with two Ca2+ ions and a monosaccharide (here fucose). The monomer is a b-sandwich composed of 8 antiparallel strands of which the five first are arranged in Greek key [5]. (MOLSCRIPT ) The principle implemented is gas phase diffusion. The protein complexed with various monosaccharides crystallises at pH 8.5 and at 20°C in the presence of ammonium sulphate at 1.75 mol/L. Crystsllisation trials of protein complexed with trisaccharides of the Lea and Lex family have started and small crystals grow in the presence of PEG 5000 and cacodylate. PAII-L/L-Fucose Binding site An increase of fucosylated oligosaccharides of Lewis type is observed, often accompanied by sialylation and/or sulphatation [1]. The recognition of these glycoconjugates present on the surface of the target cells by specialised bacterial proteins, the lectins, is of therapeutic interest. PAII-L/L-Galactose PAII-L/LewisA [4] Garber, N., Guempel, U., Gilboa-Garber, N. & Doyle, R. J. (1987) FEMS Microbiol. Lett. 48, [5] E. Mitchell C. Houles, D. Sudakevitz, M. Wimmerova, C. Gautier, S. Pérez, A. M. Wu, N. Gilboa-Garber, A. Imberty. Nature Struct. Biol. 9 (2002) [6] Bettler, E., Karababa, M., Mazeau, K., Petrova, P. & Pérez, S., in Perspectives in Structural Biology (Vijayan, M., Yathindra, N., and Kolaskar, A. S., eds) Indian Academy of Sciences and Universities Press, Hyderabad, 1999, pp Inhibition of PA-IIL binding to human blood group H and Leb active glycoprotein Inhibitor Relative potence Sialyl Lewis a Lewis a 3’Fucosyllactose L-Fucose 1 Lewis x Sialyl Lewix x The inhibitory activity was estimated from inhibition curves. Relative potency is the reciprocal of the relative inhibiting concentration when fucose was taken as 1.0 The stereochemical basis for the very high affinity of PAII-L for fucose is essentially due to the occurrence of the two calcium ions in the site. The nature of the group at C-5 of monosaccharides has no clear structural effect but modifies the thermodynamics of binding. Molecular modelling together with binding studies indicates that the lectin has higher affinity for oliogosaccharides of the Lea series. Structural analysis of the binding site gives the basis for design of high affinity ligand that could serve as anti-adhesion drugs. Conclusions Starting from the position of the fucose on the site of connection, the trisaccharides Lewis x and Lewis a were docked in their conformation of lowest energy. The geometrical optimisation of the complex, including the side chains, were calculated with the Sybyl software using the energy parameters developed for protein/carbohydrate interactions [6]. Modelling of interaction with Lewis trisaccharides Lewis x Lewis a Coordination - hydrogen bonds -(mJ) time (s) mJ/moles of ligand [ligand] Heats for each injection of ligand Microcalorimetry: thermodynamics of interactions Isothermal Titration Calorimetry (ITC) allows the determination of the thermodynamic data of interaction, i.e. the contributions of entropy (DS) and enthalpy (DH) to the free energy (DG). Other characteristics such as the affinity constant (K) as well as the stoichiometry (n) of the interaction between the PA-IIL lectin and various monosaccharides have also been determined. [1] Scharfman, A. et al. (1999) Glycobiology 9, [2] Gilboa-Garber, N., in Lectins Biology, Biochemistry, Clinical Biochemistry, Vol. 3 Bog-Hansen, T, Spengler, G. A. eds) Walter de Gruyter, Berlin, 1983, pp [3] Gilboa-Garber, N., Katcoff, D. J. & Garber, N. C. (2000) FEMS Immunol. Med. Microbiol. 29, PAII-L/L-Fuc (1.0Å) PAII-L/Me-b-D-Ara (1.8Å) PAII-L/L-Gal (1.5Å) Preliminary thermodynamics data for binding of monosacchardies to PA-IIL Data averaged on two experiments. Standard deviation is large. Crystal structures of the different complexes High resolution crystal structure Electron density map 2Fo-Fc, s=2, Fo-Fc, s =3 , s= -3