Spatial structure of plant cell wall polysaccharides and its functional significance

Plant polysaccharides comprise the major portion of organic matter in the biosphere. The cell wall built on the basis of polysaccharides is the key feature of a plant organism largely determining its biology. All together, around 10 types of polysaccharide backbones, which can be decorated by different substituents giving rise to endless diversity of carbohydrate structures, are present in cell walls of higher plants. Each of the numerous cell types present in plants has cell wall with specific parameters, the features of which mostly arise from the structure of polymeric components. The structure of polysaccharides is not directly encoded by the genome and has variability in many parameters (molecular weight, length, and location of side chains, presence of modifying groups, etc.). The extent of such variability is limited by the “functional fitting” of the polymer, which is largely based on spatial organization of the polysaccharide and its ability to form supramolecular complexes of an appropriate type. Consequently, the carrier of the functional specificity is not the certain molecular structure but the certain type of the molecules having a certain degree of heterogeneity. This review summarizes the data on structural features of plant cell wall polysaccharides, considers formation of supramolecular complexes, gives examples of tissue- and stage-specific polysaccharides and functionally significant carbohydrate-carbohydrate interactions in plant cell wall, and presents approaches to analyze the spatial structure of polysaccharides and their complexes.     

Derivatives of 6-deuterio-D-glucose,absolute configuration at C-6, and steric disposition of an acetoxymethyl group

Arabinoxylan preparations from sugar cane show temperature-induced shifts of optical rotation in aqueous methyl sulphoxide solution, the sign and magnitude of which depend on the content of arabinofuranose side-groups. The shift has a sigmoidal form and shows a distinct hysteresis loop on heating and cooling. This evidence, together with the sign and magnitude, is interpreted in terms of a conformation change of the backbone of β-(1→4)-linked D-xylose residues, from the random coil to an ordered, ribbon-like conformation similar to that which is known to exist in the solid state. The driving force for this change is not intramolecular but derives from an aggregation that occurs simultaneously and can be detected by other methods. The arabinofuranose side-groups can be incorporated into the ordered assembly and contribute to the optical rotation shift, and they therefore have an unusual role compared with other polysaccharide side-chains. We conclude that the optical rotation shifts show a “melting” and re-formation of ordered associations which may imitate the natural biological cohesion between hemicellulose chains.     

Conformational behavior of hyaluronan in relation to its physical properties as probed by molecular modeling

Hyaluronan (HA) is a linear charged polysaccharide whose structure is made up of repeating disaccharide units. Apparently conflicting reports have been published about the nature of the helical structure of HA in the solid state. Recent developments in the field of molecular modeling of polysaccharides offer new opportunities to reexamine the structural basis underlying the formation and stabilization of ordered structures and their interactions with counterions. The conformational spaces available and the low energy conformations for the disaccharide, trisaccharide, and tetrasaccharide segments of HA were investigated via molecular mechanics calculations using the MM3 force field. First, the results were used to access the configurational statistics of the corresponding polysaccharide. A disordered chain having a persistence length of 75 A at 25 degrees C is predicted. Then, the exploration of the stable ordered forms of HA led to numerous helical conformations, both left- and right-handed, having comparable energies. Several of these conformations correspond to the experimentally observed ones and illustrate the versatility of the polysaccharide. The double stranded helical forms have also been explored and theoretical structures have been compared to experimentally derived ones.     

Optical polarization reveals different ultrastructural molecular arrangement of polysaccharides in the yeast cell walls.

The topo-optical aldehyde bisulfite-toluidine blue (ABT) reaction of vicinal OH and amino-OH groups offers new ways to study the ultrastructure of polysaccharides in different biological substrates. Through oriented dye binding on the reacting groups, the ABT reaction induces strong birefringence on the linearly ordered polysaccharides, which is negative with respect to their chain length. Using this method, two types of molecular order of the polysaccharides could be distinguished in the cell walls and capsules of yeasts. (1) The optically negative spherulitic character of the yeasts after the ABT reaction indicated that the toluidine blue molecules were bound tangentially (in a surface-parallel pattern) while the polysaccharide chains of the cell walls and capsules were oriented mainly radially. This structural pattern may be explained as resulting from a helicoid conformation of the polysaccharide component. (2) Acid or alkali hydrolysis removed the radially oriented polysaccharide component of the cell wall. The remaining, resistant polysaccharides showed up in the form of optically positive spherulites indicating radially oriented dye molecules on a circularly ordered, micellar polysaccharide texture.     

Folding of polypeptide chains induced by the amino acid side-chains

Conformational calculations with the use of semi-empirical potential functions have been applied to the analysis of the folding of peptide chains. In particular, the part played by the amino acid side-chains in the adoption of folded conformations has been investigated.The results show that the preferred conformations of short peptides are mostly extended ones. However, from a given peptide chain-length, the side-chain to backbone and side-chain to side-chain interactions become strong enough so that definite sequences of amino acids can induce a transition from extended to folded conformations. We propose to call these folded structures “conformational nuclei”. The type of “nucleus” formed is dependent on both the amino acid composition and the sequence.Our results strongly support the hypothesis that folding of polypeptide chains can occur through a nucleation process that could be induced by the side-chains.     

Associations of like and unlike polysaccharides: Mechanism and specificity in galactomannans,interacting bacterial polysaccharides,and related systems

Chiroptical, rheological, and n.m.r.-relaxation evidence is presented, to identify interactions of two types between different polysaccharides: (1) mutual exclusion of incompatible molecules, with consequent increase in the effective concentration of both; and (2) energetically favourable association of structurally and sterically regular chain-segments. β-1,4-linked plant polysaccharides interact by association of unsubstituted backbone regions, either with like chians, or with sterically compatible, unlike molecules. Extracellular polysaccharides (xanthans) of Xanthomonas plant pathogens maintain their ordered native conformation in solution, and this accounts for their industrially valuable, rheological peculiarities. These materials bind strongly to the plant glycans. Random-coil bacterial gums show no such interactions, although dextran enhances autogelation of galactomannans by exclusion. Extracellular polysaccharides from Arthrobacter species also have ordered native conformations in solution, but do not share the specific interactions of xanthan. Native xanthan shows marked specificity in its interactions with plant glycans, indicating a possible biological role in host-pathogen recognition.     

Structural and chemical characterization of abienan, a pectin from the greenery of Siberian fir (Abies sibirica L.)

A pectic polysaccharide, earlier called abienan, has been extracted from the fresh greenery of Siberian fir (Abies sibirica Ledeb.) and described. Like many pectic polysaccharides, the abienan macromolecule consists of linear and branched regions. Results of the ion-exchange chromatography and partial acidolysis demonstrate that linear regions of the abienan macromolecule consist of 1,4-??-D-galacturonan. The side carbohydrate chains of branched regions consist mainly of arabinofuranose and galactopyranose residues.     

Intrinsic Disorder in the Protein Data Bank

Abstract

The Protein Data Bank (PDB) is the preeminent source of protein structural information. PDB contains over 32,500 experimentally determined 3-D structures solved using X-ray crystallography or nuclear magnetic resonance spectroscopy. Intrinsically disordered regions fail to form a fixed 3-D structure under physiological conditions. In this study, we compare the amino-acid sequences of proteins whose structures are determined by X-ray crystallography with the corresponding sequences from the Swiss-Prot database. The analyzed dataset includes 16,370 structures, which represent 18,101 PDB chains and 5,434 different proteins from 910 different organisms (2,793 eukaryotic, 2,109 bacterial, 288 viral, and 244 archaeal). In this dataset, on average, each Swiss-Prot protein is represented by 7 PDB chains with 76% of the crystallized regions being represented by more than one structure. Intriguingly, the complete sequences of only ~7% of proteins are observed in the corresponding PDB structures, and only ~25% of the total dataset have >95% of their lengths observed in the corresponding PDB structures. This suggests that the vast majority of PDB proteins is shorter than their corresponding Swiss-Prot sequences and/or contain numerous residues, which are not observed in maps of electron density. To determine the prevalence of disordered regions in PDB, the residues in the Swiss-Prot sequences were grouped into four general categories, “Observed” (which correspond to structured regions), “Not observed” (regions with missing electron density, potentially disordered), “Uncharacterized,” and “Ambiguous,” depending on their appearance in the corresponding PDB entries. This non-redundant set of residues can be viewed as a ‘fragment’ or empirical domain database that contains a set of experimentally determined structured regions or domains and a set of experimentally verified disordered regions or domains. We studied the propensities and properties of residues in these four categories and analyzed their relations to the predictions of disorder using several algorithms. “Non-observed,” “Ambiguous,” and “Uncharacterized” regions were shown to possess the amino acid compositional biases typical of intrinsically disordered proteins. The application of four different disorder predictors (PONDR® VL-XT, VL3-BA, VSL1P, and IUPred) revealed that the vast majority of residues in the “Observed” dataset are ordered, and that the “Not observed” regions are mostly disordered. The “Uncharacterized” regions possess some tendency toward order, whereas the predictions for the short “Ambiguous” regions are really ambiguous. Long “Ambiguous” regions (>70 amino acid residues) are mostly predicted to be ordered, suggesting that they are likely to be “wobbly” domains.

Overall, we showed that completely ordered proteins are not highly abundant in PDB and many PDB sequences have disordered regions. In fact, in the analyzed dataset ~10% of the PDB proteins contain regions of consecutive missing or ambiguous residues longer than 30 amino-acids and ~40% of the proteins possess short regions (≥10 and <30 amino-acid long) of missing and ambiguous residues.     

Spectroscopic characterisation of order-disorder transitions for extracellular polysaccharides of arthrobacter species

The conformational behaviour of the extracellular polysaccharides from Arthrobacter species of soil-borne bacteria has been investigated by nuclear magnetic resonance relaxation and optical rotation. Polysaccharides from A. stabilis, A. viscosus, and A. viscosus sp. n, in solution at room temperature, all show evidence of an ordered conformation which can be melted out on heating. The temperature course of this transition, however, shows considerable variation with bacterial species. Thus A. stabilis polysaccharide shows a very sharp conformational transition centred around 60°, whereas the transitions of the polysaccharides from both strains of A. viscosus occur over a much broader temperature-range. The transition for the polysaccharide of A. viscosus sp. n is again centred close to 60°, whereas, for A. viscosus, melting of the tertiary structure of the polysaccharide is incomplete at 100°. O-Deacetylation destroys the ordered conformation of both A. viscosus polysaccharides. The ordered structure of A. stabilis polysaccharide, by contrast, is stabilised by removal of acyl substituents (which here include succinic half-ester). Understanding of the conformational state of these materials affords considerable insight into their gelation behaviour and unusual solution rheology. The known solution interactions with certain plant polysaccharides suggest a possible biological role for Arthrobacter polysaccharides in relationships with components of plant root-systems.     

A novel strategy to generate biologically active neo-glycosaminoglycan conjugates

Heparin and heparan sulfate are structurally related polysaccharides with a variety of biological effects/functions. Most of these effects are due to interactions, of varying specificity, between the negatively charged polysaccharide chains and proteins. While such interactions generally involve a single saccharide domain of decasaccharide size or less, ternary complexes of two protein molecules binding to separate domains on a single polysaccharide chain are known to occur. To facilitate studies on domain organization and its importance for biological function a strategy was developed to chemically conjugate defined heparin oligomers in linear and chemoselective fashion. The procedure requires that the oligosaccharide to provide the reducing-terminal domain of the conjugate is generated by lyase degradation of a parent polysaccharide, whereas the nonreducing-terminal domain is obtained through deaminative cleavage with nitrous acid. The applicability of the method was demonstrated by constructing a conjugate composed of two heparin 12-mers, of which the reducing-terminal component contained the antithrombin-binding region, whereas the nonreducing-terminal domain did not. Contrary to any of the unconjugated oligomers, the product was found to efficiently promote the inactivation of thrombin by antithrombin.     

The crystal structure of Klebsiella pneumoniae FeoA reveals a site for protein-protein interactions

In order to establish infection, pathogenic bacteria must obtain essential nutrients such as iron. Under acidic and/or anaerobic conditions, most bacteria utilize the Feo system in order to acquire ferrous iron (Fe²⁺) from their host environment. The mechanism of this process, including its regulation, remains poorly understood. In this work, we have determined the crystal structure of FeoA from the nosocomial agent Klebsiella pneumoniae (KpFeoA). Our structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, we demonstrate that KpFeoA can assume both “open” and “closed” conformations, controlled by binding at this Leu-rich ridge. We propose a model in which a “C-shaped” clamp along the FeoA surface mediates interactions with its partner protein, FeoB. These findings are the first to demonstrate atomic-level details of FeoA-based protein-protein interactions and provide a framework for testing FeoA-FeoB interactions, which could be exploited for future antibiotic developments.     

Pectin polysaccharides of rowan Sorbus aucuparia L.

Water-soluble polysaccharide fractions were extracted from the fruit of rowan Sorbus aucuparia L. by water and 0.7% ammonium oxalate water solution. The total yield was 4.2%. It is demonstrated that these fractions are pectin polysaccharides, and their carbon chains are primarily composed of galactunoric acid residue (up to 68%), arabinose and galactose. Sephacryl S-500 gelfiltration of rowan fruit pectin polysaccharides proved their relative homogeneity pertaining to their molecular weights, whereas endo-polygalacturonase enzymatic hydrolysis gives evidence of the presence of extended galacturonan (rhamnogalacturonan) ranges in their carbohydrate chains. Methylation of rowan pectin polysaccharides shows that their carbohydrate pendants are formed by 1,5-linked arabinofuranose residue, 1,4-linked glucopyranose residue, 1,6-linked galactopyranose residue, 1,3,6-linked mannopyranose residue and 1,3,6-linked galactopyranose residue. Glucopyranose residue is identified at non reducible ends of these pendants. It was demonstrated that antioxidant activity of water solutions of pectin polysaccharides extracted from rowan S. aucuparia L. (0.5 mg/mL) is 37?C53% of trolox activity, which is 100%.     

Dissecting the conformational determinants of chitosan and chitlac oligomers

Chitosan and its highly hydrophilic 1‐deoxy‐lactit‐1‐yl derivative (Chitlac) are polysaccharides with increasing biomedical applications. Aimed to unravel their conformational properties we have performed a series of molecular dynamics simulations of Chitosan/Chitlac decamers, exploring different degrees of substitution (DS) of lactitol side chains. At low DS, two conformational regions with different populations are visited, while for DS ≥ 20% the oligomers remain mostly linear and only one main region of the glycosidic angles is sampled. These conformers are (locally) characterized by extended helical “propensities”. Helical conformations 3₂ and 2_1, by far the most abundant, only develop in the main region. The accessible conformational space is clearly enlarged at high ionic strength, evidencing also a new region accessible to the glycosidic angles, with short and frequent interchange between regions. Simulations of neutral decamers share these features, pointing to a central role of electrostatic repulsion between charged moieties. These interactions seem to determine the conformational behavior of the chitosan backbone, with no evident influence of H‐bond interactions. Finally, it is also shown that increasing temperature only slightly enlarges the available conformational space, but certainly without signs of a temperature‐induced conformational transition.     

Interactions between chondroitin-6-sulfate and poly-L-lysine in aqueous solution: Circular dichroism studies

The interactions between chondroitin-6-sulfate (chondroitin sulfate C) and poly-L -lysine have been studied as models for investigation of possible complex formation between fibrous proteins and mucopolysaccharides. Results obtained using circular dichroism spectroscopy show that poly-L -lysine adopts the α-helical conformation in dilute aqueous salt solution at pH 7 when mixed with chondroitin-6-sulfate, rather than the “charged-coil” observed in the absence of this mucopolysaccharide. This conformation-directing interaction is at a maximum when the ratio of lysine to disaccharide residues is 1 : 1. Changes in the CD spectrum of a 1 : 1 mixture following increase in the salt concentration, or addition of non-polar solvents, indicate that the interaction is ionic in nature. No such effects are observed for non-sulfated mucopolysaccharides mixed with poly-L -lysine, suggesting that, for chondroitin-6-sulfate, it is the sulfate groups rather than the carboxyls which interact with the amine groups of the polypeptide. Elevation of the temperature leads to disruption of the interactions between the polypeptide and polysaccharide species. A sharp melting transition occurs at 47.0 ± 1.0°C, when the poly-L -lysine reverts to the “charged-coil” conformation. The sharp transition suggests that regular ionic bonds are formed between the polypeptide and polysaccharide. These results suggest that a conformation-directing interaction may occur between sulfated mucopolysaccharides and the polar regions of collagen and other fibrous proteins.     

Conformational analysis of xyloglucans

Xyloglucan isolated from the elongating regions of pea stems was examined using X-ray diffraction and energy calculations. The X-ray fibre pattern suggested that the backbone (1----4)-beta-D-glucan takes an extended two-fold helix similar to common cellulose. In order to study side chains (xylosyl or fucosyl-galactosyl-xylosyl residues) of the polysaccharide, energetically preferable conformations were searched by calculation of interactions between non-bonded atom pairs. A stepwise calculation for the conformation of fucosyl-galactosyl-xylosyl residue gave 10 allowed area (phi-psi) maps which are useful to deduce xyloglucan conformations of both monocotyledons and dicotyledons in the walls of growing plant cells.     

Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases

This work describes the structure of a variety of lecithin-water phases observed below the “melting” temperature of the hydrocarbon chains, with special emphasis on the conformation of the chains. The lecithins studied in this work are the homologous series dioctanoyl to distearoyl, 2-decanoyl-1-stearoyl, and a preparation from hen eggs. The hydrocarbon chains are found to adopt a variety of conformations in addition to type α, the liquid-like organization observed above the melting temperature. Type β: the chains are stiff and parallel, oriented at right angles to the plane of the lamellae and packed with rotational disorder in a two-dimensional hexagonal lattice ($\text{a ~ 4.85}\text{A}\text{?}\text{)}$. Type β′: similar to β, but with the chains tilted with respect to the normal to the lamellae. Type δ: the chains are probably coiled into helices, whose axes are perpendicular to the plane of the polar groups and are packed with rotational disorder in a two-dimensional square lattice ($\text{a ~ 4.80}\text{A}\text{?}\text{)}$, α is the predominant conformation, common to most lipids in the presence of water and at sufficiently high temperature, and the one more relevant to membranes; β is observed at lower temperatures in lipids whose chains are heterogeneous and in the presence of very small amounts of water; β′ is found in synthetic lecithins with identical chains, in the presence of variable amounts of water; δ is observed in dry lecithins. A highly ordered crystalline phase, yet displaying rotational disorder of the chains, is observed in almost dry lecithins. Most of the phases are lamellar, and contain one lipid bilayer per repeat unit. Two phases display two-dimensional lattices: Pδ, formed by ribbon-like elements with the chains in the δ conformation; Pβ′, formed by lamellae of type β′ distorted by periodic ripples. The results emphasize the clear-cut difference between the liquid-like and the other types of partly ordered conformations, as well as the correlations which exist between the chemical composition and the structure of the lipids below the melting temperature of the chains.     

A CD study of uncoupling protein-1 and its transmembrane and matrix-loop domains

Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40-50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly beta- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1.