Disaccharide Complexes Research Articles

Influence of Association on Binding of Disaccharides to YKL-39 and hHyal-1 Enzymes.

Disaccharide complexes have been shown experimentally to be useful for drug delivery or as an antifouling surface biofilm, and are promising drug-encapsulation and delivery candidates. Although such complexes are intended for medical applications, to date no studies at the molecular level have been devoted to the influence of complexation on the enzymatic decomposition of polysaccharides. A theoretical approach to this problem has been hampered by the lack of a suitable computational tool for binding such non-covalent complexes to enzymes. Herein, we combine quantum-mechanical calculations of disaccharides complexes with a nonstandard docking GaudiMM engine that can perform such a task. Our results on four different complexes show that they are mostly stabilized by electrostatic interactions and hydrogen bonds. This strong non-covalent stabilization demonstrates the studied complexes are some excellent candidates for self-assembly smart materials, useful for drug encapsulation and delivery. Their advantage lies also in their biocompatible and biodegradable character.

Biomolecular condensation via ultraviolet excitation in vacuo

Recently, we reported that 157nm vacuum ultraviolet irradiation (VUV) of proton-bound peptide dimers trapped in a vacuum, results in the elimination of water and formation of a peptide bond [J. Am. Chem. Soc. 133 (2011) 15834–15837]. Here, we further explore the ability to form a covalent bond between biomolecular ions with photoexcitation. Photoexcitation of long-lived charge-bound complexes appears to be a general phenomenon, resulting in the loss of water and the formation of covalent bonds between many types of molecules. Several examples are described, including: the linear coupling of amino acid chains that produce octapeptides from tetrapeptide complexes; inter-molecular cross-linking of amino acid side chains, and glycosidic bond formation between disaccharide complexes. Simple mechanisms for each case are proposed.

Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate–lysozyme interaction studies

By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using (1)H saturation transfer difference (STD) effects and transferred (1)H,(1)H-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides β-d-Galp-(1→4)-β-D-GlcpNAc-OMe and β-D-GlcpNAc-(1→4)-β-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/π-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on (1)H NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.

Influence of glycosidic linkage on the nature of carbohydrate binding in β-prism I fold lectins: An X-ray and molecular dynamics investigation on banana lectin–carbohydrate complexes

The three crystal structures reported here provide details of the interactions of mannose and the mannosyl-α-1,3-mannose component of a pentamannose with banana lectin and evidence for the binding of glucosyl-α-1,2-glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-β-1,3-glucose. Modeling studies on the three disaccharide complexes with the reducing end and the nonreducing end at the primary binding site are also provided here. The results of the X-ray and modeling studies show that the disaccharides with an α-1,3 linkage prefer to have the nonreducing end at the primary binding site, whereas the reducing end is preferred at the site when the linkage is β-1,3 in mannose/glucose-specific β-prism I fold lectins. In the corresponding galactose-specific lectins, however, α-1,3-linked disaccharides cannot bind the lectin with the nonreducing end at the primary binding site on account of steric clashes with an aromatic residue that occurs only when the lectin is galactose-specific. Molecular dynamics simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin.

Structure and sugar-specificity of basic winged-bean lectin: structures of new disaccharide complexes and a comparative study with other known disaccharide complexes of the lectin

Crystal structures of the complexes of basic winged-bean agglutinin with the disaccharides Galalpha1-4Gal (galabiose), Galalpha1-6Glc (mellibiose) and Galalpha1-4Galbeta-Et have been determined and the complex with Galalpha1-2Gal has been modelled. The interactions of the nonreducing Gal with the lectin at the primary site are the same as those in the known complexes with disaccharides having the alpha1-->3 linkage. The second residue in Galalpha1-4Gal and Galalpha1-6Glc forms a water bridge to the lectin, while the ethyl group in Galalpha1-4Galbeta-Et makes nonpolar interactions. In complexes involving disaccharides with alpha1-3 linkages, which form part of the A and B blood-group substances, the second sugar residue forms a direct hydrogen bond to the variable loop in the binding site of the lectin. This in part explains the specificity of the lectin for the blood-group substances and also the higher affinity of alpha1-->3-linked disaccharides for the lectin compared with disaccharides involving other linkages. Including those reported here, 14 crystal structures involving the lectin, accounting for 54 crystallographically independent subunits, are available. A comparative study of these structures shows that the region involving the curved beta-sheet which nestles the metal ions is relatively rigid. The carbohydrate-binding region is perched on this region. The flat beta-sheet, which is involved in oligomerization and exhibits considerable variability in legume lectins, is relatively flexible. Indeed, the structures of basic winged-bean lectin have been of critical importance in establishing legume lectins as a family of proteins in which small alterations in essentially the same tertiary structure lead to large variations in quaternary association. They have also provided a structural explanation of the blood-group specificity of the lectin.

Peanut agglutinin, a lectin with an unusual quaternary structure and interesting ligand binding properties

Lectins are multivalent proteins which play their biological role through the ability to specifically bind different carbohydrate structures. This ability has also led to their myriad applications. They occur in all forms of life. Among plant lectins, those from leguminous plants constitute the most thoroughly studied family. Most of the well-characterized legume lectins can be classified as mannose (Man)/glucose (Glc) specific or galactose (Gal)/N-acetylgalactosamine (GalNAc) specific. Tetrameric, non-glycosylated peanut agglutinin (PNA) is the most thoroughly investigated member of the Gal/GalNAc specific family of legume lectins. Its structure indicated that open quaternary association also needed to be considered when dealing with multimeric proteins. The structure also helped to establish legume lectins as a family of proteins in which small alterations in essentially the same tertiary structure lead to large changes in quaternary association. It provides an explanation for the exclusive specificity of PNA for galactose at the primary site. The structures of the relevant complexes demonstrated that the specificity of the lectin for T-antigen at the disaccharide level is caused by a water-bridge, thus establishing water-mediated interactions as a strategy for generating ligand specificity. In disaccharide complexes, the second residue interacts directly with the protein only when the glycosidic linkage is β1-3 or β1-4. Studies on the interactions of PNA with a bivalent ligand provide valuable insights into the multivalency of lectins. The availability of detailed structural information on the lectin permitted rational re-design of its combining site. A comparison of the available structural data on PNA led to the elucidation of the limited plasticity of the lectin molecule and its relation to function. An exploration of its hydration shell brought out the importance of conserved water molecules in the structure and function of PNA. Interestingly, while the Man/Glc specific concanavalin A, the most thoroughly studied lectin to date, represents the conventional wisdom on legume lectins, PNA, perhaps the next most extensively studied legume lectin, largely highlights departures from it.

Differential Recognition of the Type I and II H Antigen Acceptors by the Human ABO(H) Blood Group A and B Glycosyltransferases

The human ABO(H) blood group A and B antigens are generated by the homologous glycosyltransferases A (GTA) and B (GTB), which add the monosaccharides GalNAc and Gal, respectively, to the cell-surface H antigens. In the first comprehensive structural study of the recognition by a glycosyltransferase of a panel of substrates corresponding to acceptor fragments, 14 high resolution crystal structures of GTA and GTB have been determined in the presence of oligosaccharides corresponding to different segments of the type I (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->3)-beta-D-GlcNAcp-OR, where R is a glycoprotein or glycolipid in natural acceptors) and type II (alpha-l-Fucp-(1-->2)-beta-D-Galp-(1-->4)-beta-d-GlcNAcp-OR) H antigen trisaccharides. GTA and GTB differ in only four "critical" amino acid residues (Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268). As these enzymes both utilize the H antigen acceptors, the four critical residues had been thought to be involved strictly in donor recognition; however, we now report that acceptor binding and subsequent transfer are significantly influenced by two of these residues: Gly/Ser-235 and Leu/Met-266. Furthermore, these structures show that acceptor recognition is dominated by the central Gal residue despite the fact that the L-Fuc residue is required for efficient catalysis and give direct insight into the design of model inhibitors for GTA and GTB.

Toward the understanding of the structure and dynamics of protein–carbohydrate interactions: molecular dynamics studies of the complexes between hevein and oligosaccharidic ligands

Herein we study, through all atom molecular dynamics simulations, the complex between hevein and two N-acetylated chitin oligomers, namely N, N ′-diacetylchitobiose and N, N ′, N ″-triacetylchitotriose. The results of the simulations for two disaccharide complexes and one trisaccharide complex show that a carbohydrate oligomer is able to move on the surface of the relatively flat binding pocket of hevein, therefore occupying different binding subpockets. Statistical analysis methods were also applied in order to define the principal overall motions in the complexes, showing how the different ligands in the simulations modulate the protein motions. The oligosaccharide binding can be considered as defined by a subtle balance between enthalpic (formation of intermolecular interactions between the ligand and the receptor) and entropic (due mainly to the possibility for the sugar to move on the surface of the protein domain) effects, determining multiple binding conformations. This structural and dynamical view could parallel the results obtained by regularly used restrained MD simulations based on NOE NMR data that provide a well defined structure for both the disaccharide and trisaccharide complexes, and agrees with the observations for longer oligosaccharide chains.

Thermochemistry and structures of Na + coordinated mono- and disaccharide stereoisomers

The Na + affinities of several mono- and disaccharide stereoisomers are determined in the gas phase based on the dissociations of Na +-bound heterodimers [saccharide + B i ]Na +, where B i represents a reference base of known Na + affinity (kinetic method). The compounds investigated include the pentoses arabinose, xylose, and ribose; the hexoses glucose, galactose, and mannose; and the disaccharides melibiose, gentiobiose, and lactose. The decompositions of [saccharide + B i ]Na + are assessed as a function of internal energy, to thereby obtain both absolute Na + affinities as well as relative entropies of Na + attachment. The Na + affinities measured are consistent with multidentate coordination of sodium ion by the oxygen sites of the saccharides. In general, hexoses bind Na + stronger than pentoses, suggesting that the hydroxymethyl substituent equips them with more conformational flexibility and larger inductive effects for complexing Na +. The latter properties are further enhanced in the disaccharides, which also carry more basic substituents; as a result, disaccharides form even stronger bonds to Na +. The entropies of Na + attachment are found to rise in the order pentose < hexose < disaccharide, pointing to an increase in this direction of the rotational flexibility lost after attachment of Na +. The favored [monosaccharide + Na] + structures predicted computationally contain pyranose rings in chair or boat conformations that permit tri- or tetradentate Na + coordination and hydrogen bonds between the hydroxyl ligands; the most stable disaccharide complexes are tetradentate and involve chair forms. In the calculated structures, the pyranose O atom and the hydroxymethyl group(s) generally participate in the Na + binding, in agreement with the experimental trends. Small changes in the saccharide stereochemistry alter the optimum Na + coordination possible and, therefore, the Na + affinity; as a result, the latter thermochemical property is ideally suitable for the distinction of stereoisomeric saccharides.

Thermodynamics of bovine spleen galectin-1 binding to disaccharides: correlation with structure and its effect on oligomerization at the denaturation temperature.

Isothermal titration calorimetry (ITC) measurements of the binding 1-beta carbohydrate-substituted galactopyranoside derivatives to galectin-1 from bovine spleen, a dimer with one binding site per subunit, were performed at 283-285 and 298 K. The disaccharides were lactose, methyl beta-lactoside, lactulose, 4-O-beta-D-galactopyranosyl-D-mannopyranoside, 3-O-beta-D-galactopyranosyl-D-arabinose, 2'-O-methyllactose, lacto-N-biose, N-acetyllactosamine, and thiodigalactopyranoside. The site binding enthalpies, DeltaHb, are the same at both temperatures and range from -42.2 +/- 3.3 kJ mol-1 for thiodigalactopyranoside to -24.5 +/- 0.5 kJ mol-1 for lacto-N-biose, and the site binding constants range from 4.86 +/- 0.78 x 10(3) M-1 for methyl beta-lactoside at 297.8 K to 6.54 +/- 0.97 x 10(4) M-1 for N-acetyllactosamine at 281.3 K. The binding reactions are enthalpically driven, exhibit enthalpy-entropy compensation, and, with the exception of N-acetyllactosamine, follow a van't Hoff dependence of the binding constant on temperature. The number of contacts at distances <4.0 A between the disaccharide and galectin was determined from the energy-minimized conformation of the complex derived from the X-ray crystallographic structure of the galectin-N-acetyllactosamine complex determined by Liao et al. [Liao, D. I., Kapadia, G., Ahmed, H., Vasta, G. R., and Herzberg, O. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1428-1432]. The binding enthalpies calculated from changes in the solvent-accessible surface areas of the galectin binding site upon binding of the disaccharide were in close agreement with the experimental values for lactose, lactulose, lacto-N-biose, and N-acetyllactosamine, all of which exhibit binding enthalpies >-36 kJ mol-1. Differential scanning calorimetry measurements on solutions of galectin and its disaccharide complexes show that the galectin dimer does not dissociate upon denaturation in contrast to the legume lectins. At the denaturation temperature, the galectin in the absence of sugar exists as a tetramer, and the extent of this association is substantially reduced in the presence of a disaccharide.

The 2.0 å structure of a cross-linked complex between snowdrop lectin and a branched mannopentaose: evidence for two unique binding modes

Background Galanthus nivalis agglutinin (GNA), a mannose-specific lectin from snowdrop bulbs, is a tetrameric member of the family of Amaryllidaceae lectins that exhibit antiviral activity towards HIV. Its subunits are composed of three pseudo-symmetrically related β sheet domains, each with a conserved mannose-binding site. Crystal structures of monosaccharide and disaccharide complexes of GNA have revealed that all 12 binding sites of the tetramer are functional, and that the degree of occupancy is dependent on the availability of subsidiary interactions from neighboring subunits. The complex of GNA with a branched mannopentaose ((Man α1,6-( α1,3-Man)Man- α1,6-( α1,3-Man)Man) described here simulates a more biologically relevant complex. Results Two unique mannopentaose binding modes co-exist in the tetragonal structure (1 subunit/asymmetric unit) of the complex. In one, the conserved monosaccharide-binding pocket in domain 1 (CRD 1) is utilized for cross-linkage of twofold related GNA dimers by the outer 3,6 tri-Man arm, which alternates between two orientations consistent with crystal symmetry. Inter-linked dimers assemble helically along the 4 1 crystal axis forming a pore-like structure. In the second binding mode, the complete 3,6 tri-Man arm binds to an extended binding region in domain 3 (CRD 3) with subsites for each terminal Man and the internal Man positioned in the conserved monosaccharide pocket. The two remaining mannose residues are not visible in either binding mode. Conclusions This structure provides insights into possible mechanisms of the cross-linkage that is known to occur when lectins interact with specific multivalent cell surface receptors during events such as agglutination and mitogenic stimulation. By virtue of the large number of sites available for mannose binding, GNA has multiple possibilities of forming unique lattice structures. The two distinctly different binding modes observed in this study confirm that high affinity mannose binding occurs only at the two domain sites located near dimer interfaces.

Differentiation of stereochemistry of glycosidic bond configuration: Tandem mass spectrometry of diastereomeric cobalt-glucosyl-glucose disaccharide complexes

Configurations of glycosidic linkages (α or β) in a series of 1,3-, 1,4-, and 1,6-glucosyl-glucose disaccharides were differentiated by tandem mass spectrometry. Diastereomeric octahedral complexes, [Co(+3) (acac)2/disaccharide](+), were generated in situ via fast-atom bombardment ionization. Mass-analyzed, ion kinetic energy spectra of the metastable complexes obtained in the absence of collision gas indicated that the major product ion results from the loss of an acetylacetonate ligand, which thus generates the ion [Co(+2)(acac)/disaccharide](+). Kinetic energy release measurements for this dissociation display a consistently greater value for complexes that possess an α-linked disaccharide relative to those that possess β-linked disaccharides, regardless of linkage position.

Transition-metal-saccharide chemistry: synthesis and characterisation of D-galactose, D-fructose, D-glucose, D-xylose, D-ribose, and maltose complexes of Mn(II)

Monosaccharide ( D-Gal, D-Fru, D-Glc, D-Xyl, D-Rib) and disaccharide ( D-Mal) complexes of Mn(II) were synthesised from non-aqueous solutions using [NEt 4] 2[MnCl 2Br 2] and MnCl 2 · 4H 2O. The isolated and purified complexes were characterised by diffuse reflectance, FTIR, EPR, DTA, TGA, magnetic susceptibility, and cyclic voltammetry techniques. Synthesis from [NEt 4] 2[MnCl 2Br 2] yielded dimeric complexes 1–3 and 9, and from MnCl 2 · 4H 2O it yielded trimeric complexes 4–8. These water-soluble complexes were found to be anionic with a Mn-to-saccharide ratio of 1:1 for monosaccharide complexes 1–8 and 2:1 for disaccharide complex 9. The complexes were found to be hydrolytically stable in solution at higher concentrations (⩾ 4 mM), as studied by cyclic voltammetry.

Metal-saccharide chemistry and biology: saccharide complexes of zinc and their effect on metallothionein synthesis in mice

Monosaccharide ( d-Fru, d-Gal, d-Glc, d-Xyl, and d-Rib) and disaccharide (Mal) complexes of Zn 2+ were synthesised using different precursors and isolated in the solid state. These were found to be anionic with a Zn-to-saccharide ratio of 1:1 and 2:1 for monosaccharide and disaccharide complexes, respectively. Electrochemical behaviour in aqueous solution was studied by extensive cyclic voltammetric studies in the pH range 3.7–10.3. The effect of subcutaneously injected Zn- d-Fru, Zn- d-Gal and Zn- d-Glc complexes on the metallothionein synthesis in mice was found to be significant in the liver, but not in the brain.

Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin.

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.