Molecular arrangement between multivalent glycocluster and Pseudomonas aeruginosa LecA (PA-IL) by atomic force microscopy: influence of the glycocluster concentration.

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA-IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA-IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra-galactosylated glycocluster and the PA-IL lectin for high concentration of lectins (2.5 µM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one-dimensional elongated aggregates to two-dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction.

Oligosaccharide structures: theory versus experiment.

Recently, the interdependency of theoretical and experimental approaches in the structure determination of oligosaccharides has been confirmed. More accurate simulations are possible because of the advances in software and computers. Meanwhile, improvements in NMR techniques permit the measurement of numerous structural and dynamical parameters, either for the free state or for carbohydrate ligands bound to receptors. Several crystal structures of isolated or protein-complexed oligosaccharides give new clues for modeling the intermolecular forces that drive the interactions.

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.

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 basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.

T4 phage beta-glucosyltransferase: substrate binding and proposed catalytic mechanism.

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 which catalyses the transfer of glucose (Glc) from uridine diphosphoglucose (UDP-Glc) to 5-hydroxymethylcytosine (5-HMC) in double-stranded DNA. The glucosylation of T4 phage DNA is part of a phage DNA protection system aimed at host nucleases. We previously reported the first three-dimensional structure of BGT determined from crystals grown in ammonium sulphate containing UDP-Glc. In this previous structure, we did not observe electron density for the Glc moiety of UDP-Glc nor for two large surface loop regions (residues 68-76 and 109-122). Here we report two further BGT co-crystal structures, in the presence of UDP product (form I) and donor substrate UDP-Glc (form II), respectively. Form I crystals are grown in ammonium sulphate and the structure has been determined to 1.88 A resolution (R -factor 19.1 %). Form II crystals are grown in polyethyleneglycol 4000 and the structure has been solved to 2.3 A resolution (R -factor 19.8 %). The form I structure is isomorphous to our previous BGT UDP-Glc structure. The form II structure, however, has allowed us to model the two missing surface loop regions and thus provides the first complete structural description of BGT. In this low-salt crystal form, we see no electron density for the Glc moiety from UDP-Glc similar to previous observations. Biochemical data however, shows that BGT can cleave UDP-Glc in the absence of DNA acceptor, which probably accounts for the absence of Glc in our UDP-Glc substrate structures. The complete BGT structure now provides a basis for detailed modelling of a BGT HMC-DNA ternary complex. By using the structural similarity between the catalytic core of glycogen phosphorylase (GP) and BGT, we have modelled the position of the Glc moiety in UDP-Glc. From these two models, we propose a catalytic mechanism for BGT and identify residues involved in both DNA binding and in stabilizing a "flipped-out" 5-HMC nucleotide.

Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus.

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104. Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.

Multivalent thioglycopeptoids via photoclick chemistry: potent affinities towards LecA and BC2L-A lectins.

Solution-phase synthesis of linear and cyclic ß- and α,ß-peptoids was coupled to photo-induced thiol-ene coupling reaction to readily access multivalent thioglycoclusters. A tetrameric cyclic ß-peptoid scaffold displaying 1-thio-ß-d-galactose or 1-thio-α-d-mannose has revealed by ITC experiments efficient binding potency for bacterial lectins LecA and BC2L-A, respectively.

The interplay of autophagy and ß-Catenin signaling regulates differentiation in acute myeloid leukemia.

The major feature of leukemic cells is an arrest of differentiation accompanied by highly active proliferation. In many subtypes of acute myeloid leukemia, these features are mediated by the aberrant Wnt/ß-Catenin pathway. In our study, we established the lectin LecB as inducer of the differentiation of the acute myeloid leukemia cell line THP-1 and used it for the investigation of the involved processes. During differentiation, functional autophagy and low ß-Catenin levels were essential. Corresponding to this, a high ß-Catenin level stabilized proliferation and inhibited autophagy, resulting in low differentiation ability. Initiated by LecB, ß-Catenin was degraded, autophagy became active and differentiation took place within hours. Remarkably, the reduction of ß-Catenin sensitized THP-1 cells to the autophagy-stimulating mTOR inhibitors. As downmodulation of E-Cadherin was sufficient to significantly reduce LecB-mediated differentiation, we propose E-Cadherin as a crucial interaction partner in this signaling pathway. Upon LecB treatment, E-Cadherin colocalized with ß-Catenin and thereby prevented the induction of ß-Catenin target protein expression and proliferation. That way, our study provides for the first time a link between E-Cadherin, the aberrant Wnt/ß-Catenin signaling, autophagy and differentiation in acute myeloid leukemia. Importantly, LecB was a valuable tool to elucidate the underlying molecular mechanisms of acute myeloid leukemia pathogenesis and may help to identify novel therapy approaches.

Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases.

Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.

Single-coordinate-driving method for molecular docking: application to modeling of guest inclusion in cyclodextrin.

An extension of the computer program CICADA has been developed that allows us to use the single-coordinate-driving (SCD) method for flexible molecular docking. The docking procedure is composed of three independent space rotations, three independent translations, and the torsions selected by the user. One of the coordinates is driven; the other coordinates are relaxed. This procedure follows low-energy wells on the potential energy surface of the entire system. The program allows us to dock more than one ligand molecule to the receptor. We ran two test examples, docking N,N-dimethylformamide into alpha-cyclodextrin and R-phenoxypropionic acid into beta-cyclodextrin. The test examples showed that the SCD approach is able to overcome high-energy barriers and to cover the entire box within which the search is performed. The limitations of molecular dynamics docking in comparison with our approach also are discussed. The philosophy of the newly developed approach is not only to find the best dock for the receptor-ligand(s) system, but also to describe all the important binding modes and provide a good starting point for studying the dynamics within the cavity during the docking process.

Conformational analysis of biantennary glycans and molecular modeling of their complexes with lentil lectin.

Some mannose-binding legume lectins show higher affinity for fucosylated glycans than for glycans without fucose. These lectins possess a secondary binding site. Owing to the possibility of additional fucose binding, oligosaccharides adopt different conformations depending on whether they contain fucose or not. To study these conformational differences, complexes of fucosylated and unfucosylated glycans with Lens culinaris lectin have been modeled. Starting points were X-ray structures of lentil lectin and complexes of the homologous Lathyrus ochrus lectin. The SYBYL molecular modeling package with the TRIPOS force field was used. Two different models were built, displaying in both a network of hydrogen bonds between the saccharide and the binding site. Furthermore, to compare the free and bound ligand, conformational analysis in the free state has been performed. A complete analysis of all possible disaccharide fragments has been performed using the MM3 force field. A CICADA analysis employing the same force field was carried out to study the complete oligosaccharide. Low-energy conformers found by CICADA were clustered in conformational families and analyzed in terms of flexibility and rotational barriers. All values of glycosidic torsion angles are in the range as calculated by MM3 for the disaccharides.

X-ray structure determination and modeling of the cyclic tetrasaccharide cyclo.

The cyclic tetrasaccharide cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] is the major compound obtained by the action of endo-alternases on the alternan polysaccharide. Crystals of this cyclo-tetra-glucose belong to the orthorhombic space group P2(1)2(1)2(1) with a = 7.620(5), b = 12.450(5) and c = 34.800(5) A. The asymmetric unit contains one tetrasaccharide together with five water molecules. The tetrasaccharide adopts a plate-like overall shape with a very shallow depression on one side. The shape is not fully symmetrical and this is clearly apparent on comparing the (phi, psi) torsion angles of the two alpha-(1-->6) linkages. There is almost 10 degrees differences in phi and more than 20 degrees differences in psi. The hydrogen bond network is asymmetric, with a single intramolecular hydrogen bond: O-2 of glucose ring 1 being the donor to O-2 of glucose ring 3. These two hydroxyl groups are located below the ring and their orientation, dictated by this hydrogen bond, makes the floor of the plate. Among the five water molecules, one located above the center of the plate occupies perfectly the shallow depression in the plate shape formed by the tetrasaccharide. Molecular dynamics simulation of the tetrasaccharide in explicit water allows rationalization of the discrepancies observed between the X-ray structures and data obtained previously by NMR.

Predicting helical structures of the exopolysaccharide produced by Lactobacillus sake 0- 1.

The viscous exopolysaccharide (EPS) produced by Lactobacillus sake 0- 1 is a high molecular mass polymer (Mm 6 x 10(6) Da) consisting of pentasaccharide repeating units with a composition of D-glucose, L-rhamnose, and sn-glycerol 3-phosphate in molar ratios of 3:2:1. One of the rhamnose residues in the repeating unit is partially 2-O-acetylated. The O-deacetylated, deglycerophosphorylated EPS has been investigated by molecular mechanics calculations. A complete conformational analysis of each of the constituent disaccharide fragments has been performed using the flexible residue approach with the MM3(92) force field. Furthermore, using the same force field, CICADA analyses were accomplished on hexa- and octasaccharide substructure of the polysaccharide. Based on these analyses, insight was obtained into nine conformational minima for the polysaccharide. The low energy conformations found by CICADA were extrapolated to regular polysaccharide structures using a polysaccharide builder program. The generated helices exhibit either 2-fold or 3- or 4-fold right-handed chiralities, and in each case the helices are highly extended.

Solvent-dependent conformational behaviour of lipochitoligosaccharides related to Nod factors.

The solution conformation of two lipooligosaccharides related to Nod factors or lipochitoligosaccharides have been analysed by 1D and 2D 1H and 13C NMR spectroscopy, molecular mechanics and dynamics calculations. The obtained data indicate that the glycosidic torsion angles have restricted fluctuations, but may adopt a variety of shapes. Remarkably, the relative orientation of the fatty acid chain towards the oligosaccharide backbone is solvent dependent. In water solution, the acyl residue and the oligosaccharide adopt a quasi-parallel orientation for a significant amount of time.

Conformational analysis and molecular modelling of the branching point of amylopectin.

The conformational properties of 6(2) alpha-D-glucosylmaltotriose have been studied using energy calculations that include van der Waals interactions, hydrogen bond stabilization, exo-anometric effect and torsional potential contributions. The calculations focused mainly on the conformational properties displayed at the alpha(1----6) linkage within the tetrasaccharide for which the conformational space is reported. The tetrasaccharide molecule was then considered as a model compound of the branching point in amylopectin. From molecular modelling, some basic structural features associated with branching were clearly established. It was found that, among the low energy arrangements, the side chain would fold back onto the main backbone, thereby producing dense three-dimensional structures in which a 'parallel' arrangement is achieved. The branching between two strands of the double helix, as found in the crystalline moiety of A and B starches, was further investigated. It was found that one particular set of conformations about the glycosidic linkages in the two different strands, could result in an arrangement such that strands could be connected through an alpha(1----6) glycosidic linkage, with a minimum of distortion. The three-dimensional features derived from the molecular modelling agree with the physical properties and mode of biogenesis within the starch granule; they are in accord with a 'cluster' type of structure.

Flexibility in a tetrasaccharide fragment from the high mannose type of N-linked oligosaccharides.

An analysis has been carried out of the three-dimensional structure of a tetrasaccharide, Man(alpha 1-3)Man(alpha 1-6)Man(beta 1-4)GlcN Ac beta 1-OCD3, which is a fragment from the high mannose type of N-linked oligosaccharides. Although earlier work had suggested that this fragment might adopt a stable three-dimensional structure, both n.m.r. and conformational energy calculations support the existence of an ensemble of structures. The conformational entropy calculated from the ensemble and the distribution of distances between the terminal Man(alpha 1-3) and GlcN Ac residues, however, suggests that a significant fraction of the ensemble has the two terminal residues in close proximity.

Internal motion in carbohydrates as probed by n.m.r. spectroscopy.

In the present study a combination of proton and carbon relaxation rates have been measured for several oligosaccharides at different temperatures. Correlation times, which have been calculated from both sets of data, have been compared in an attempt to establish the relative rate of internal motion. All the data suggest that these motions are not slow with respect to the overall tumbling.

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.