Anne Imberty

The double-helical nature of the crystalline part of A-starch.

A new three-dimensional structure of the crystalline part of A-starch is described in which the unit cell contains 12 glucose residues located in two left-handed, parallel-stranded double helices packed in a parallel fashion; four water molecules are located between these helices. Chains are crystallized in a monoclinic lattice with a = 2.124 nm, b = 1.172 nm, c = 1.069 nm and gamma = 123.5 degrees, the c axis being parallel to the helix axis. Systematic absences are consistent with the space group B2. The structure was derived from joint use of electron diffraction of single crystals, X-ray powder patterns decomposed into individual peaks and previously reported X-ray fibre diffraction data after adequate re-indexing. The repeating unit consists of a maltotriose moiety where the glucose residues have the 4C1 pyranose conformation and are alpha(1----4) linked. The conformation of the glycosidic linkage is characterized by torsion angles (phi, psi) which take the values (91.8, -153.2), (85.7, -145.3) and 91.8, -151.3); all the primary hydroxyl groups exist in a gauche-gauche conformation. There are no intramolecular hydrogen bonds. Within the double helix, interstrand stabilization is achieved without any steric conflict and through the occurrence of O(2)...O(6) type hydrogen bonds. The present structure is consistent with both physicochemical and biochemical aspects of the crystalline component of the cereal starch granules.

Structural diversity of heparan sulfate binding domains in chemokines

Heparan sulfate (HS) molecules are ubiquitous in animal tissues where they function as ligands that are dramatically involved in the regulation of the proteins they bind. Of these, chemokines are a family of small proteins with many biological functions. Their well-conserved monomeric structure can associate in various oligomeric forms especially in the presence of HS. Application of protein surface analysis and energy calculations to all known chemokine structures leads to the proposal that four different binding modes are created by the folding and oligomerization of these proteins. So, based on the present state of our knowledge, four different clusters of amino acids should be involved in the recognition process. Our results help to rationalize how unique sequences of HS specifically bind any given chemokine. The conclusions open the route for a rational design of compounds of therapeutical interest that could influence chemokine activity.

Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients. The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure. Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate–protein interactions. Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Lea) series may be the preferred ligands of this lectin. Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.

Glycomimetics and glycodendrimers as high affinity microbial anti-adhesins.

Adhesion to epithelial surface is often the first step in bacterial and viral infection. In this process, the microbes use a variety of proteins for interaction with host carbohydrates presented as glycoconjugates on cell surfaces. Crystal structures of adhesin and lectin binding sites in complexes with oligosaccharide open the route for design and synthesis of glycomimetics, glycodendrimers, and glycopolymers that are able to block infection at an early stage.

Microbial recognition of human cell surface glycoconjugates

Infection by pathogens is generally initiated by the specific recognition of host epithelia surfaces and subsequent adhesion is essential for invasion. In their infection strategy, microorganisms often use sugar-binding proteins, that is lectins and adhesins, to recognize and bind to host glycoconjugates where sialylated and fucosylated oligosaccharides are the major targets. The lectin/glycoconjugate interactions are characterized by their high specificity and most of the time by multivalency to generate higher affinity of binding. Recent crystal structures of viral, bacterial, and parasite receptors in complex with human histo-blood group epitopes or sialylated derivatives reveal new folds and novel sugar-binding modes. They illustrate the tight specificity between tissue glycosylation and lectins.

STRUCTURE/FUNCTION STUDIES OF GLYCOSYLTRANSFERASES

Glycosyltransferases are the enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. The analysis of the wealth of sequences that are now available in databases allowed the determination of conserved peptide motifs for each class of enzyme. Recent experimental data demonstrated their importance in donor and acceptor substrate binding and in catalysis. Fold-recognition studies provided the first models of the catalytic domains of some of these enzymes, while the first successes in glycosyltransferase crystallography are opening new routes in structural glycobiology.

Characterization of the stromal cell-derived factor-1alpha-heparin complex.

The binding of chemokines to glycosaminoglycans is thought to play a crucial role in chemokine functions. It has recently been shown that stromal cell-derived factor-1α (SDF-1α), a CXC chemokine with potent anti-human immunodeficiency virus activity, binds to heparan sulfate through a typical consensus sequence for heparin recognition (BBXB, where B is a basic residue KHLK, amino acids 24–27). Calculation of the accessible surface, together with the electrostatic potential of the SDF-1α dimer, revealed that other amino acids (Arg-41 and Lys-43) are found in the same surface area and contribute to the creation of a positively charged crevice, located at the dimer interface. GRID calculations confirmed that this binding site will be the most energetically favored area for the interaction with sulfate groups. Site-directed mutagenesis and surface plasmon resonance-based binding assays were used to investigate the structural basis for SDF-1α binding to heparin. Among the residues clustered in this basic surface area, Lys-24 and Lys-27 have dominant roles and are essential for interaction with heparin. Amino acids Arg-41 and Lys-43 participate in the binding but are not strictly required for the interaction to take place. Direct binding assays and competition analysis with monoclonal antibodies also permitted us to show that the N-terminal residue (Lys-1), an amino acid critical for receptor activation, is involved in complex formation. Binding studies with selectively desulfated heparin, heparin oligosaccharides, and heparitinase-resistant heparan sulfate fragments showed that a minimum size of 12–14 monosaccharide units is required for efficient binding and that 2-O- andN-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1α, and a docking study was undertaken. During the energy minimization process, heparin lost its perfect ribbon shape and fitted the protein surface perfectly. In the model, Lys-1, Lys-24, Lys-27, and Arg-41 were found to have the major role in binding a polysaccharide fragment consisting of 13 monosaccharide units.

Heparan Sulfate/Heparin Oligosaccharides Protect Stromal Cell-derived Factor-1 (SDF-1)/CXCL12 against Proteolysis Induced by CD26/Dipeptidyl Peptidase IV

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that is constitutively expressed in most tissues and displayed on the cell surface in association with heparan sulfate (HS). Its numerous biological effects are mediated by a specific G protein-coupled receptor, CXCR4. A number of cells inactivate SDF-1 by specific processing of the N-terminal domain of the chemokine. In particular, CD26/dipeptidyl peptidase IV (DPP IV), a serine protease that co-distributes with CXCR4 at the cell surface, mediates the selective removal of the N-terminal dipeptide of SDF-1. We report here that heparin and HS specifically prevent the processing of SDF-1 by DPP IV expressed by Caco-2 cells. The level of processing increases with the level of differentiation of these cells, which correlates with an increase of DPP IV activity. A mutant SDF-1 that does not interact with HS is readily cleaved by DPP IV, a process that is not inhibited by HS, demonstrating that a productive interaction between HS and SDF-1 is required for the protection to take place. Moreover, we found that protection depends on the degree of polymerization of the HS sulfated S-domains. Finally a structural model of SDF-1, in complex with HS oligosaccharides of defined length, rationalizes the experimental data. The mechanisms by which HS regulates SDF-1 may thus include, in addition to its ability to locally concentrate the chemokine at the cell surface, a control of selective protease cleavage events that directly affect the chemokine activity.

A new bioinformatic approach to detect common 3D sites in protein structures.

An innovative bioinformatic method has been designed and implemented to detect similar three‐dimensional (3D) sites in proteins. This approach allows the comparison of protein structures or substructures and detects local spatial similarities: this method is completely independent from the amino acid sequence and from the backbone structure. In contrast to already existing tools, the basis for this method is a representation of the protein structure by a set of stereochemical groups that are defined independently from the notion of amino acid. An efficient heuristic for finding similarities that uses graphs of triangles of chemical groups to represent the protein structures has been developed. The implementation of this heuristic constitutes a software named SuMo (Surfing the Molecules), which allows the dynamic definition of chemical groups, the selection of sites in the proteins, and the management and screening of databases. To show the relevance of this approach, we focused on two extreme examples illustrating convergent and divergent evolution. In two unrelated serine proteases, SuMo detects one common site, which corresponds to the catalytic triad. In the legume lectins family composed of >100 structures that share similar sequences and folds but may have lost their ability to bind a carbohydrate molecule, SuMo discriminates between functional and non‐functional lectins with a selectivity of 96%. The time needed for searching a given site in a protein structure is typically 0.1 s on a PIII 800MHz/Linux computer; thus, in further studies, SuMo will be used to screen the PDB. Proteins 2003;52:137–145.

Role of LecA and LecB Lectins in Pseudomonas aeruginosa-Induced Lung Injury and Effect of Carbohydrate Ligands

ABSTRACT Pseudomonas aeruginosa is a frequently encountered pathogen that is involved in acute and chronic lung infections. Lectin-mediated bacterium-cell recognition and adhesion are critical steps in initiating P. aeruginosa pathogenesis. This study was designed to evaluate the contributions of LecA and LecB to the pathogenesis of P. aeruginosa-mediated acute lung injury. Using an in vitro model with A549 cells and an experimental in vivo murine model of acute lung injury, we compared the parental strain to lecA and lecB mutants. The effects of both LecA- and Lec B-specific lectin-inhibiting carbohydrates (α-methyl-galactoside and α-methyl-fucoside, respectively) were evaluated. In vitro, the parental strain was associated with increased cytotoxicity and adhesion on A549 cells compared to the lecA and lecB mutants. In vivo, the P. aeruginosa-induced increase in alveolar barrier permeability was reduced with both mutants. The bacterial burden and dissemination were decreased for both mutants compared with the parental strain. Coadministration of specific lectin inhibitors markedly reduced lung injury and mortality. Our results demonstrate that there is a relationship between lectins and the pathogenicity of P. aeruginosa. Inhibition of the lectins by specific carbohydrates may provide new therapeutic perspectives.