Insights into stereochemical features of sulphated carbohydrates: X-ray crystallographic and modelling investigations

The three-dimensional structures of the 2-, 3-, 4- and 6-monosulphatesof methyl -D-galactopyranoside have been determined by X-raycrystallography; the first two as the sodium salt, the thirdas both the sodium and potassium salts, and the fourth as apotassium salt. These represent the principal sulphated monomersof the carrageenan polysaccharides. The results extend our knowledgeof the stereochemical features, such as ring conformation, sulphategeometry, hydrogen bonding and cation co-ordination, which characterizesulphated monosaccharides. The stereochemical data have beenused to derive a mean geometry of the O-sulphate group and aset of force constants for use in molecular mechanics calculationson sulphated monosaccharides. These may be used in an extrapolationof the populations of stable conformers of related oligo- andpolysaccharides. crystal structures molecular mechanics O-sulphated carbohydrates X-ray diffraction     

A molecular builder for carbohydrates: Application to polysaccharides and complex carbohydrates

A new procedure (POLYS) for producing three-dimensional structures of polysaccharides and complex carbohydrates is described. This employs a builder concept combining a database of monosaccharide structures with a database containing information on populations of independent neighboring glycosidic linkages in disaccharide fragments. The computer program is written in C, and it can cope with both the complexity and the diversity of carbohydrates and the unique topological features arising from multiple branching. A simple ASCII syntax was developed for describing the primary structures in accordance with IUPAC nomenclature. The translation of the primary structure is made through the combined use of a lexical analyzer and a command interpreter. In this way the program can be considered as a compiler of primary structures of carbohydrates. However, it also generates secondary and tertiary structures in the form of Cartesian coordinates in formats used by most molecular mechanics programs and packages. In our laboratory POLYS was exhaustively tested on standard homopolysaccharide systems such as cellulose and mannan and found to work very well. We now report the ease of use and the efficiency of the molecular builder in applications to more complex carbohydrate systems. These include the structural exploration of a pentaantennary oligosaccharide having 135 residues, the complex family of pectic polysaccharides including the organization and distribution of side chains (arabinan, arabinogalactan, and galactan) on the rhamnogalacturonan backbone. © 1996 John Wiley & Sons, Inc.     

Structural glycobiology: a game of snakes and ladders

Oligo- and polysaccharides are infamous for being extremely flexible molecules, populating a series of well-defined rotational isomeric states under physiological conditions. Characterization of this heterogeneous conformational ensemble has been a major obstacle impeding high-resolution structure determination of carbohydrates and acting as a bottleneck in the effort to understand the relationship between the carbohydrate structure and function. This challenge has compelled the field to develop and apply theoretical and experimental methods that can explore conformational ensembles by both capturing and deconvoluting the structural and dynamic properties of carbohydrates. This review focuses on computational approaches that have been successfully used in combination with experiment to detail the three-dimensional structure of carbohydrates in a solution and in a complex with proteins. In addition, emerging experimental techniques for three-dimensional structural characterization of carbohydrate-protein complexes and future challenges in the field of structural glycobiology are discussed. The review is divided into five sections: (1) The complexity and plasticity of carbohydrates, (2) Predicting carbohydrate-protein interactions, (3) Calculating relative and absolute binding free energies for carbohydrate-protein complexes, (4) Emerging and evolving techniques for experimental characterization of carbohydrate-protein structures, and (5) Current challenges in structural glycoscience.     

Solution NMR conformation of glycosaminoglycans

Nuclear magnetic resonance (NMR) spectroscopy has been giving a pivotal contribution to the progress of glycomics, mostly by elucidating the structural, dynamical, conformational and intermolecular binding aspects of carbohydrates. Particularly in the field of conformation, NOE resonances, scalar couplings, residual dipolar couplings, and chemical shift anisotropy offsets have been the principal NMR parameters utilized. Molecular dynamics calculations restrained by NMR-data input are usually employed in conjunction to generate glycosidic bond dihedral angles. Glycosaminoglycans (GAGs) are a special class of sulfated polysaccharides extensively studied worldwide. Besides regulating innumerous physiological processes, these glycans are also widely explored in the global market as either clinical or nutraceutical agents. The conformational aspects of GAGs are key regulators to the quality of interactions with the functional proteins involved in biological events. This report discusses the solution conformation of each GAG type analyzed by one or more of the above-mentioned methods.     

Crystallography of polysaccharides: Current state and challenges

Polysaccharides are the most abundant class of biopolymers, holding an important place in biological systems and sustainable material development. Their spatial organization and intra- and intermolecular interactions are thus of great interest. However, conventional single crystal crystallography is not applicable since polysaccharides crystallize only into tiny crystals. Several crystallographic methods have been developed to extract atomic-resolution structural information from polysaccharide crystals. Small-probe single crystal diffractometry, high-resolution fiber diffraction and powder diffraction combined with molecular modeling brought new insights from various types of polysaccharide crystals, and led to many high-resolution crystal structures over the past two decades. Current challenges lie in the analysis of disorder and defects by further integrating molecular modeling methods for low-resolution diffraction data.     

The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.     

Crystal structure of a pro-inflammatory lectin from the seeds of Dioclea wilsonii Standl

The crystal structure and pro-inflammatory property of a lectin from the seeds of Dioclea wilsonii (DwL) were analyzed to gain a better understanding of structure/function relationships of Diocleinae lectins. Following crystallization and structural determination by standard molecular replacement techniques, DwL was found to be a tetramer based on PISA analysis, and composed by two metal-binding sites per monomer and loops which are involved in molecular oligomerization. DwL presents 96% and 99% identity with two other previously described lectins of Dioclea rostrata (DRL) and Dioclea grandiflora (DGL). DwL differs structurally from DVL and DRL with regard to the conformation of the carbohydrate recognition domain and related biological activities. The structural analysis of DwL in comparison to other Diocleinae lectins can be related to the differences in the dose-dependent pro-inflammatory effect elicited in Wistar rats, probably via specific interactions with mast cells complex carbohydrate, resulting in significant paw edema. DwL appears to be involved in positive modulation of mast cell degranulation via recognition of surface carbohydrates. Since this recognition is dependent on site volume and CRD configuration, edematogenesis mediated by resident cells varies in potency and efficacy among different Diocleinae lectins.     

Adulteration identification of some fungal polysaccharides with SEM, XRD, IR and optical rotation: A primary approach

Edible mushrooms have been consumed in Asian countries for healthy functions historically with polysaccharides, which have been manufactured as ending products, as one of the major bioactive components. Driven by profits, some inglorious manufacturers conducted adulteration using cheap carbohydrates. The extracted polysaccharides from Agaricus blazei Murill, Ganoderma lucidum and Lentinus edodes were chosen as products and maltodextrin, carboxymethyl cellulose (CMC), water soluble corn starch and guar gum as adulterants for adulteration investigation. Scanning electron microscope (SEM) observation implied regular granular shapes of fungal polysaccharides different significantly from the adulterants. Infrared spectra revealed α- and β-anomeric types of polysaccharides and carbohydrates. X-ray diffractometry (XRD) suggested different diffraction patterns of polysaccharides from adulterant carbohydrates, the former were amorphous while the later somewhat crystalline of different crystallinity. Chiral properties of polysaccharides and carbohydrates showed big differences in specific rotation with good correlation between the content of “pure” fungal polysaccharides and adulterants.     

A unique structural pattern shared by T-cell-activating and abscess-regulating zwitterionic polysaccharides

In contrast to the conventional dogma that carbohydrates are poorly immunogenic T-cell-independent antigens, zwitterionic polysaccharides (ZPSs) can significantly stimulate T-cell proliferation and regulate abscess formation in bacterial infection. Despite their similar biological activities, ZPSs from various bacteria are greatly different in primary chemical compositions and building block linkages. To identify the common structural features that govern the peculiar immunologic activity of ZPSs, we have been determining three-dimensional structures of compositionally different ZPSs by NMR spectroscopy and molecular mechanics and dynamics calculations. We report here the conformation of type 1 capsular polysaccharide from the human pathogen Streptococcus pneumoniae (Sp1) to be a right-handed helix with repeated zwitterionically charged grooves. We also report the striking similarity between the structures of Sp1 and our previously determined PS A2 from Bacteroides fragilis. These results support our hypothesis that T-cell-activating ZPSs assume similar conformational and charge patterns that are recognized by specific receptors and that account for their common property as T-cell activators.     

Core oligosaccharide structure from the highly phytopathogenic Agrobacterium tumefaciens TT111 and conformational analysis of the putative rhamnan epitope

The structure of the complex mixture of the core oligosaccharide components of the lipooligosaccharide (LOS) fraction of Agrobacterium tumefaciens strain TT111 was determined directly on the deacetylated products by means of spectroscopical methods. The rhamnan oligosaccharide elongating the inner Kdo residue shares structural features with other polysaccharides from well-known plant pathogenic bacteria. Its conformation was determined through extensive molecular dynamic (MD) analysis and presents an epitope similar to that recognized from the plant defense system.     

Surprising roles of electrostatic interactions in DNA-ligand complexes

The positions of cations in x-ray structures are modulated by sequence, conformation, and ligand interactions. The goal here is to use x-ray diffraction to help resolve structural and thermodynamic roles of specifically localized cations in DNA-anthracycline complexes. We describe a 1.34 A resolution structure of a CGATCG(2)-adriamycin(2) complex obtained from crystals grown in the presence of thallium (I) ions. Tl(+) can substitute for biological monovalent cations, but is readily detected by distinctive x-ray scattering, obviating analysis of subtle differences in coordination geometry and x-ray scattering of water, sodium, potassium, and ammonium. Six localized Tl(+) sites are observable adjacent to each CGATCG(2)-adriamycin(2) complex. Each of these localized monovalent cations are found within the G-tract major groove of the intercalated DNA-drug complex. Adriamycin appears to be designed by nature to interact favorably with the electrostatic landscape of DNA, and to conserve the distribution of localized cationic charge. Localized inorganic cations in the major groove are conserved upon binding of adriamycin. In the minor groove, inorganic cations are substituted by a cationic functional group of adriamycin. This partitioning of cationic charge by adriamycin into the major groove of CG base pairs and the minor groove of AT base pairs may be a general feature of sequence-specific DNA-small molecule interactions and a potentially useful important factor in ligand design.     

Interaction of substrate uridyl 3'',5''-adenosine with ribonuclease A: a molecular dynamics study.

A wealth of information available from x-ray crystallographic structures of enzyme-ligand complexes makes it possible to study interactions at the molecular level. However, further investigation is needed when i) the binding of the natural substrate must be characterized, because ligands in the stable enzyme-ligand complexes are generally inhibitors or the analogs of substrate and transition state, and when ii) ligand binding is in part poorly characterized. We have investigated these aspects in the binding of substrate uridyl 3',5'-adenosine (UpA) to ribonuclease A (RNase A). Based on the systematically docked RNase A-UpA complex resulting from our previous study, we have undertaken a molecular dynamics simulation of the complex with solvent molecules. The molecular dynamics trajectories of this complex are analyzed to provide structural explanations for varied experimental observations on the ligand binding at the B2 subsite of ribonuclease A. The present study suggests that B2 subsite stabilization can be effected by different active site groups, depending on the substrate conformation. Thus when adenosine ribose pucker is O4'-endo, Gln69 and Glu111 form hydrogen-bonding contacts with adenine base, and when it is C2'-endo, Asn71 is the only amino acid residue in direct contact with this base. The latter observation is in support of previous mutagenesis and kinetics studies. Possible roles for the solvent molecules in the binding subsites are described. Furthermore, the substrate conformation is also examined along the simulation pathway to see if any conformer has the properties of a transition state. This study has also helped us to recognize that small but concerted changes in the conformation of the substrate can result in substrate geometry favorable for 2',3' cyclization. The identified geometry is suitable for intraligand proton transfer between 2'-hydroxyl and phosphate oxygen atom. The possibility of intraligand proton transfer as suggested previously and the mode of transfer before the formation of cyclic intermediate during transphosphorylation are discussed.     

Structure and interactions in simple solutions

Neutron scattering with hydrogen/deuterium isotopic substitution techniques has been used to investigate the full range of structural interactions in a dilute 0.02 mol fraction solution of tertiary butanol in water, both in the absence and in the presence of a small amount of sodium chloride. Emphasis is given to the detailed pictures of the intermolecular interactions that have been derived using the empirical potential structure refinement technique. Analysis has been performed to the level of the spatial density distribution functions that illustrate the orientational dependence of the intermolecular interactions between all combinations of molecular and ionic components. The results show the key structural motifs involved in the interactions between the various components in a complex aqueous system. They underline the structural versatility of the water molecule in accommodating a range of different kinds of interactions while retaining its characteristic first-neighbour interaction geometry. Within this framework, the results highlight the complex interplay between the polar, non-polar and charged molecular interactions that exist in the system.     

Solution and gelling properties of polysaccharide polyelectrolytes.

Attention is focussed on the special class of polysaccharide polyelectrolytes belonging to the family of the microbial polysaccharides. But a few exceptions, these are high molecular weight water- soluble polycarboxylates with complex, regular structures. Complexity and regularity in primary structure--two features normally not shared by other polysaccharides of either vegetal or animal origin--quite naturally entail unique conformational propensities, special physico-chemical properties in bulk and in solution and, as a consequence, make these biopolymers of particular interest from both a basic research and an industrial standpoint. What is outlined in this presentation should demonstrate that for many exocellular microbial polycarboxylates the solution properties are, as expected, dictated by the conformation assumed by the polyelectrolytic chains which, in turn, are governed by several free energy terms in particular stemming from specific solvent-chain interactions, among which the coulombic contribution may play a minor role.     

Role for fucose-sulfate-rich carbohydrates in the penetration of zona-pellucida-free hamster eggs by hamster spermatozoa

The interaction of acrosome-reacted hamster spermatozoa and zona-pellucida-free hamster eggs was investigated by incubating gametes in the presence of a variety of simple and complex carbohydrates. Significant inhibition of gamete fusion was achieved only in the presence of fucoidan and ascophyllin, two algal polysaccharides containing fucose sulfate. These compounds did not interfere with sperm motility, capacitation, or acrosome reactions. It is concluded that these two compounds share common structural features with putative cell-surface carbohydrates involved in sperm-oolemmal interaction.     

Glycosaminoglycan origin and structure revealed by multivariate analysis of NMR and CD spectra

Principal component analysis (PCA) is a method of simplifying complex datasets to generate a lower number of parameters, while retaining the essential differences and allowing objective comparison of large numbers of datasets. Glycosaminoglycans (GAGs) are a class of linear sulfated carbohydrates with diverse sequences and consequent complex conformation and structure. Here, PCA is applied to three problems in GAG research: (i) distinguishing origins of heparin preparations, (ii) structural analysis of heparin derivatives, and (iii) classification of chondroitin sulfates (CS). The results revealed the following. (i) PCA of heparin (13)C NMR spectra allowed their origins to be distinguished and structural differences were identified. (ii) Analysis of the information-rich (1)H and (13)C NMR spectra of a series of systematically modified heparin derivatives uncovered underlying properties. These included the presence of interactions between residues, providing evidence that a degree of degeneracy exists in linkage geometry and that a different degree of variability exists for the two types of glycosidic linkage. The relative sensitivity of each position (C or H nucleus) in the disaccharide repeating unit to changes in O-, N-sulfation and N-acetylation was also revealed. (iii) Analysis of the (1)H NMR and CD spectra of a series of CS samples from different origins allowed their structural classification and highlighted the power of employing complementary spectroscopic methods in concert with PCA.     

Comprehensive conformational study of key interactions involved in zearalenone complexation with beta-D-glucans

The beta-D-glucans from the cell wall of Saccharomyces cerevisiae have shown in vitro affinity for zearalenone. For this reason, their utilization as dietary adsorbent, to reduce the bioavailability of zearalenone, is of practical interest. Our study used powerful devices to elucidate the spatial conformation and molecular sites of interaction between ZEN and beta-D-glucans. In this respect, 1H NMR spectroscopy implicated the hydroxyl groups of the phenol moiety of zearalenone in the complexation by laminarin, a pure beta-(1,3)-D-glucan. X-ray diffraction determined that laminarin displays the conformation of a single-helix with six beta-D-glucopyranose residues per turn. At this stage, molecular modeling was useful to locate the interaction sites and to propose highly probable complexes of zearalenone with laminarin fragment. Interestingly, the beta-(1,3)-D-glucan chain favors a very stable intra-helical association with zearalenone, nicely stabilized by beta-(1,6)-D-glucans side chains. Both hydrogen bonds and van der Waals interactions were precisely identified in the complex and could thus be proposed as driving interactions to monitor the association between the two molecules.     

Influence of the membrane surface on glycolipid conformation and dynamics. An interpretation of NMR results using conformational energy calculations.

Glycolipids constitute an important class of biomolecules that are involved in biomolecular recognition. The importance of carbohydrate head group conformation in such processes is well recognized. Glycolipids typically occur as minor components of the complex heterogeneous matrix of a biological membrane. As a result, the membrane surface may not only influence head group conformation but also serves as a spatial frame in which the glycolipid is oriented and recognized. In this study, conformational energy calculations have been used to assess the conformational space available to the glucose head group of 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) in a liquid-crystalline membrane matrix. 2H NMR quadrupolar splittings are calculated and compared with those observed experimentally. This study demonstrates the importance of including surface interactions when considering the conformational space accessible to cell surface carbohydrates. The empirical approach taken here provides considerable insight at the molecular level, and offers the possibility of exploring even more complex systems.