Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

Conformation of the O-specific polysaccharide of Shigella dysenteriae type 1: molecular modeling shows a helical structure with efficient exposure of the antigenic determinant alpha-L-Rhap-(1-->2)-alpha-D-Galp.

The O-specific polysaccharide of Shigella dysenteriae type 1, which has the repeating tetrasaccharide unit -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-alpha-D-GlcNAcp-(1--> (A-B-C-D), is a major virulence factor, and it is believed that antibodies against this polysaccharide confer protection to the host. The conformational properties of fragments of this O-antigen were explored using systematic search with a modified HSEA method (GLYCAN) and with molecular mechanics MM3(96). The results show that the alpha-D-Gal-(1-->3)-alpha-D-GlcNAc linkage adopts two favored conformations, phi/psi approximately equal to -40 degrees /-30 degrees (I) and approximately 15 degrees /30 degrees (II), whereas the other glycosidic linkages only have a single favored phi/psi conformational range. MM3 indicates that the trisaccharide B-C-D and tetrasaccharides containing the B-C-D moiety exist as two different conformers, distinguished by the conformations I and II of the C-D linkage. For the pentasaccharide A-B-C-D-A' and longer fragments, the calculations show preference for the C-D conformation II. These results can explain previously reported nuclear magnetic resonance data. The pentasaccharide in its favored conformation II is sharply bent, with the galactose residue exposed at the vertex. This hairpin conformation of the pentasaccharide was successfully docked with the binding site of a monoclonal IgM antibody (E3707 E9) that had been homology modeled from known crystal structures. For fragments made of repetitive tetrasaccharide units, the hairpin conformation leads to a left-handed helical structure with the galactose residues protruding radially at the helix surface. This arrangement results in a pronounced exposure of the galactose and also the adjacent rhamnose in each repeating unit, which is consistent with the known role of the as alpha-L-Rhap-(1-->2)-alpha-D-Galp moiety as a major antigenic epitope of this O-specific polysaccharide.     

The Conformational Properties of the Glc3Man Unit Suggest Conformational Biasing within the Chaperone-assisted Glycoprotein Folding Pathway

A major puzzle is: are all glycoproteins routed through the ER calnexin pathway irrespective of whether this is required for their correct folding? Calnexin recognizes the terminal Glcα1-3Manα linkage, formed by trimming of the Glcα1-2Glcα1-3Glcα1-3Manα (Glc₃Man) unit in Glc₃Man₉GlcNAc₂. Different conformations of this unit have been reported. We have addressed this problem by studying the conformation of a series of N-glycans; i.e. Glc₃ManOMe, Glc₃Man₄,₅,₇GlcNAc₂ and Glc₁Man₉GlcNAc₂ using 2D NMR NOESY, ROESY, T-ROESY and residual dipolar coupling experiments in a range of solvents, along with solution molecular dynamics simulations of Glc₃ManOMe. Our results show a single conformation for the Glcα1-2Glcα and Glcα1-3Glcα linkages, and a major (65%) and a minor (30%) conformer for the Glcα1-3Manα linkage. Modeling of the binding of Glc₁Man₉GlcNAc₂ to calnexin suggests that it is the minor conformer that is recognized by calnexin. This may be one of the mechanisms for controlling the rate of recruitment of proteins into the calnexin/calreticulin chaperone system and enabling proteins that do not require such assistance for folding to bypass the system. This is the first time evidence has been presented on glycoprotein folding that suggests the process may be optimized to balance the chaperone-assisted and chaperone-independent pathways.     

Molecular dynamics simulation of Lewis blood groups and related oligosaccharides.

Molecular dynamics simulations without explicit inclusion of solvent molecules have been performed to study the motions of Lewisa and Lewisb blood group oligosaccharides, and two blood group A tetrasaccharides having type I and type II core chains. The blood group H trisaccharide has also been studied and compared with the blood group A type II core chain. The potential energy surface developed by Rasmussen and co-workers was used with the molecular mechanics code CHARMM. The lowest energy minima of the component disaccharide fragments were obtained from conformational energy mapping. The lowest energy minima of these disaccharide fragments were used to build the tri- and tetrasaccharides that were further minimized before the actual heating/equilibration and dynamics simulations. The trajectories of the disaccharide fragments, e.g., Fuc alpha- (1----4)GlcNAc, Gal beta-(1----4)GlcNAc, etc., show transitions among various minima. However, the oligosaccharides were found to be dynamically stable and no transitions to other minimum energy conformations were observed in the time series of the glycosidic dihedral angles even during trajectories as long as 300 ps. The stable conformations of the glycosidic linkages in the oligosaccharides are not necessarily the same as the minimum energy conformation of the corresponding isolated disaccharides. The average fluctuations of the glycosidic angles in the oligosaccharides were well within the range of +/- 15 degrees. The results of these trajectory calculations were consistent with the relatively rigid single-conformation models derived for these oligosaccharides from 1H-nmr data.     

Effect of glycosylation on structure and dynamics of MHC class I glycoprotein: a molecular dynamics study

Complex carbohydrates linked to glycoproteins are recently being implicated to play a variety of biological roles. The lack of well-resolved crystallographic coordinates of the carbohydrates makes it difficult to assess the contributions of the glycan chain on protein structure and dynamics. We have modeled two different oligosaccharides NeuNAc2Gal3Man3GlcNAc5Fuc and Man3GlcNAc4 to generate two glycosylation variants of major histocompatibility complex (MHC) class I glycoprotein. Molecular dynamics simulations of the isolated fourteen- and seven-residue oligosaccharides have been done in vacuo and in solution. The dynamics of the two glycoforms of MHC class I protein have been simulated in solution in the free as well as in the peptide-bound form. Good agreement between the calculated solution conformations of the oligosaccharides in isolated and conjugated forms and the average conformations obtained from x-ray or NMR data was observed for most of the glycosidic linkages. These molecular dynamics simulations of the isolated glycan chains and the glycoconjugates reveal the details of the conformational flexibility of the glycan chains; they also provide atomic level details of protein-carbohydrate interactions and the effect of the ligand binding on the carbohydrate structure and dynamics. It was found that though there is some flexibility in some of the glycosidic linkages in the isolated oligosaccharides, in the protein-conjugated form the linkages adopt more restricted conformations. The glycan chains protrude out into the solvent and might hinder the lateral association of the proteins. The presence of the bulky glycan chains does not affect the average backbone fold of the protein but induces local changes in protein structure and dynamics. It has been noted that the extent of the changes depends upon the nature of the attached glycan chain. The glycan chains do not appear to influence the peptide binding property of the protein directly, but may stabilize the protein residues that are involved in ligand binding.     

A designed chimeric cyanovirin‐N homolog lectin: Structure and molecular basis of sucrose binding

The NMR and X‐ray structures of a designed chimeric cyanovirin‐N homolog (CVNH) protein were determined. The individual halves of the structure are similar to their counterparts in the parent proteins, with domains A and B resembling the structures of TbCVNH and NcCVNH, respectively. No significant differences between the solution and crystal conformations were observed, although details in loop conformations and distinct crystal packing‐induced features are present. Carbohydrate binding studies by NMR revealed affinity and specificity for Glcα(1‐2)Frc and Manα(1‐2)Man, and the parental half that is devoid of any sucrose affinity in NcCVNH was transformed into a genuine sucrose binding site in the context of the chimera. The atomic details of sugar recognition are seen in the crystal structure of the protein with two bound Glcα(1‐2)Frc molecules. Both sugars exhibit different conformations around the glycosidic bond and engage in unique hydrogen bonding networks in the two sites. Although the protein is able to bind two Manα(1‐2)Man molecules, a property associated with HIV‐inactivation, no anti‐HIV activity was observed for the hybrid protein. These results provide the structural basis for sugar recognition in the CVNH family and aid in deciphering the relationship between sugar binding and anti‐HIV activity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A conformational study of the xyloglucan oligomer, XXXG, by NMR spectroscopy and molecular modeling

A structural study of the XXXG xyloglucan heptasaccharide (X = alpha-D-Xylp(1 --> 6)-beta-D-Glcp and G = beta-D-Glcp) isolated from apple fruit has been undertaken with nmr and molecular mechanics methods. Quantitative 400 MHz nmr data including nuclear Overhauser effect spectroscopy (NOESY) volumes were recorded at both 6 and 20 degrees C. In spite of severe overlapping of resonances, it was possible to estimate summed NOEs for the majority of the anomeric and glucosyl methylene protons. An ensemble-average population of preferred geometries has been established with the CICADA conformational searching algorithm associated with the MM3 force field. Comparison of the theoretical data obtained by back-calculation of the NOESY volumes from the ensemble-average distance matrix program and motional models based on the Stokes-Einstein-Debye relation satisfactorily reproduce the experimental data. Conformational averaging about the mainchain glycosidic linkages includes both the syn and anti conformers and a minor gauche-gauche population is highly probable. The theoretical data overestimate the syn preference of the Glc(c) --> Glc(b) linkage as well as the Glc(c) GT rotamer population. Finally, both the motional models and the conformational search indicate a fairly rigid backbone and greater flexiblity for the xylose side chains.     

Molecular modeling of a disialylated monofucosylated biantennary glycan of theN-acetyllactosamine type

The conformations of a disialylated monofucosylated biantennary glycan of theN-acetyllactosamine type were analysed using the Tripos 5.3 force field from the Sybyl software currently used for molecular modelling. The conformation of each glycosidic linkage was calculated when included in oligosaccharide structures of up to 5 units and the influence of the glycosidic environment on the overall structure was measured. The study clearly shows that the conformation of a branched glycan cannot result from the simple addition of the different low energy conformers of each of the glycosidic linkages constituting the glycan structure. The asymmetrical conformation of the two antennae was demonstrated. The lowest energy conformations of the overall glycan structure were built and classified into 5 main models: the Y, T, bird and broken wing conformations already described and a new one called the back folded wing conformation.     

Conformational studies on a unique bis-sulfated glycolipid using NMR spectroscopy and molecular dynamics simulations.

The time-averaged solution conformation of a unique bis-sulfated glycolipid (HSO3)2-2,6Manalpha-2Glcalpha-1-sn-2,3-O-alkylglycerol , was studied in terms of the torsional angles of two glycosidic linkages, phi (H1-C1-O-Cx) and psi (C1-O-Cx-Hx), derived from heteronuclear three-bond coupling constants (3JC,H), and inter-residual proton-proton distances from J-HMBC 2D and ROESY experiments, respectively. The dihedral angles of Glcalpha1Gro in glycolipids were determined for the first time. The C1-C4 diagonal line of the alpha-glucose ring makes an angle of approximately 120 degrees with the glycerol backbone, suggesting that the alpha-glucose ring is almost parallel to the membrane surface in contrast with the perpendicular orientation of the beta-isomer. Furthermore, minimum-energy states around the conformation were estimated by Monte Carlo/stochastic dynamics (MCSD) mixed-mode simulations and the energy minimization with assisted model building and energy refinement (AMBER) force field. The Glcalpha1Gro linkage has a single minimum-energy structure. On the other hand, three conformers were observed for the Manalpha2Glc linkage. The flexibility of Manalpha2Glc was further confirmed by the absence of inter-residual hydrogen bonds which were judged from the temperature coefficients of the chemical shifts, ddelta/dT (-10-3 p.p.m. degrees C-1), of hydroxy protons. The conformational flexibility may facilitate interaction of extracellular substances with both sulfate groups.     

Molecular structures for microbial polysaccharides. X-ray diffraction from the capsular polysaccharide of Klebsiella K-type 5

Fibre Type X-ray diffraction patterns have been obtained from oriented, semicrystalline films prepared from the sodium salt form of the capsular polysaccharide of K5. The molecule has a linear trisaccharide repeating sequence containing a 1,3 linked β-d-glucuronic acid and a 1,4 linked β-d-glucose residue, resulting in a backbone linkage geometry of Man(1 eq-4e1)-GIcUA (1-eg-4eg) Gle (1 eq-3 eq) Man. It also contains an O-acetyl group and two charged groups, namely a uronic acid and a pyruvate. Analysis of the diffraction results gives rise to an extended two-fold helical conformation with an axially projected advance of 1.35 nm which correlates directly with the covalent repeating sequence. X-ray diffraction patterns from preparations of deacetylated K5 polysaccharide showed similar conformations for the individual helices but the interchain packing arrangements are different. In each case, isolated helices have been computer generated using molecular model building procedures and the most favourable conformations for both preparations were those which contained three stabilizing interresidue hydrogen bonds, one across each of the glycosidic linkages.     

Molecular modeling of sialyloligosaccharide fragments into the active site of influenza virus N9 neuraminidase

Molecular modeling studies have been carried out to investigate the interactions between substrate sialyloligosaccharide (SOS) fragments bearing different glycosidic linkages and influenza virus N9 neuraminidase, a surface glycoprotein of influenza virus subtype N9. The studies revealed that the allowed orientation for sialic acid (SA) is less than 1% in the Eulerian space at the active site. The active site of this enzyme has enough space to accommodate various SOS fragments, NeuNAcalpha(2-3)Gal, NeuNAcalpha(2-6)Gal, NeuNAcalpha(2-8)NeuNAc and NeuNAcalpha(2-9)NeuNAc, but on specific conformations. In the bound conformation, among these substrates there exists a conformational similarity leading to a structural similarity, which may be an essential requirement for the cleavage activity of the neuraminidases irrespective of the type of glycosidic linkage.     

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.     

SWEET - WWW-based rapid 3D construction of oligo- and polysaccharides.

SUMMARY: SWEET is a WWW-based tool which rapidly converts the commonly used carbohydrate sequence information directly into a preliminary but reliable 3D model which can be visualised and written to files in several ways. AVAILABILITY: SWEET is accessible via the Internet at http://www.dkfz-heidelberg.de/spec/. CONTACT: a. bohne@dkfz-heidelberg.de or w.vonderlieth@ dkfz-heidelberg.de SUPPLEMENTARY INFORMATION: The current version of SWEET generates only one conformation out of a manifold. Several authors have analysed possible conformations of high-mannose N-linked glycans using a combination of NMR methods and computational approaches showing that such molecules are rather flexible populating normally several conformations for each glycosidic linkage. The displayed model exhibits for all glycosidic linkages a conformation which is in accordance with the reported variations of Phi, psi and omega values for specific linkage (see http://www.dkfz-heidelberg. de/spec/sweet2/doc/input/sba_example.html).     

Measuring the magnitude of internal motion in a complex hexasaccharide

For the development of a scheme for quantitative experimental estimation of internal motion in the complex human milk hexasaccharide lacto‐N‐di‐fuco hexose I (LNDFH I), we measured a large number of experimental residual dipolar couplings in liquid crystal orienting media. We present a total of 40 ¹³C? ¹H and ¹H? ¹H dipolar coupling values, each representing distinct directions of internuclear vectors. The NMR data were interpreted with established methods for analysis of rigid subdomains of the oligosaccharide as well as a novel method in which dipolar couplings were calculated over an ensemble of conformers from a solvent Molecular Dynamics trajectory using multiple linear regression analysis. The Lewis^b epitope region of LNDFH I assumed a single unique conformation with internal motion described by fluctuations of 5–10° in glycosidic dihedral angles consistent with previous studies. Greater flexibility was observed for the remaining GlcNAc1→3‐β‐D ‐Gal and β‐D ‐Gal1→4Glc linkages, with the former glycosidic linkage existing in a conformational exchange among three states. The results were also supported by similar results of calculations carried out with conformers obtained from a simple Monte Carlo simulation without explicit solvent. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 39–50, 2011.     

Molecular dynamics simulations of cyclohenicosakis-[(1-->2)-beta-D-gluco-henicosapyranosyl], a cyclic (1-->2)-beta-D-glucan (a 'cyclosophoraose') of DP 21

We report molecular dynamics simulations of cyclohenicosakis-[(1-->2)-beta-D-gluco-henicosapyranosyl], termed 'cyclosophohenicosamer', a member of a class of cyclic (1-->2)-beta-D-glucans ('cyclosophoraoses'). Our goals were to provide insights into the conformational preferences of these cyclosophoraoses. Simulated annealing and constant-temperature molecular dynamics calculations were performed on the DP 21 cyclosophohenicosamer. The radius of gyration (R(G)) of the molecule and the conformation of glycosidic dihedral angles were used to analyze the result of computational studies. Most glycosidic linkages were concentrated in the lowest-energy region of the phi-psi energy map, and the values of radius of gyration from our simulations were consistent with the reported experimental value. The simulations produced various types of compact and asymmetric conformations within reasonable ranges of the glycosidic linkage conformation and radius of gyration. The results indicate the presence of a high degree of molecular flexibility of cyclosophohenicosamer and suggest the uniqueness of inclusion complexation with other molecules through this molecular flexibility.     

Molecular dynamics simulations of a monofucosylated biantennary glycan of the N-acetyllactosamine type: the human lactotransferrin glycan.

Molecular dynamics simulations were carried out to explore the conformational flexibility of the antennae of N-linked glycans. They were performed over 200 ps in vacuo on the complete disialylated monofucosylated biantennary glycan of the N-acetyllactosaminic type. Starting from a bird-conformation, the 3-D-structure evolved through 9 successive transitional states to a new, compact and energetically favorable conformation which had never been previously described. In this conformation, both antennae are organized in two coplanar loops rolled in a contrary direction and oriented perpendicularly to the plane of the di-N-acetyl chitobiose residue leading to a 'lobster conformation'. All the glycosidic linkages of the disialylated monofucosylated biantennary glycan, except the Fuc(alpha 1-6)GlcNAc(beta 1-), were modified by a phase transition. Particularly, the Man(beta 1-4) GlcNAc(beta 1-) linkage, which was previously described by NMR and X-ray diffraction as a rigid one, was involved in numerous conformational changes during 83 ps, even before the first transition phase. The freedom of mobility of the torsional angles of the Man(alpha 1-6)Man(beta 1-) linkage was limited, under these simulation conditions, to the angle psi which took three values: 30 degrees, 90 degrees and 180 degrees. Moreover, from 150 ps up to the end of the simulation, the value of the torsional angle omega of the NeuAc(alpha 2-6)Gal(beta 1-) linkage of the alpha-1,6-antenna continuously swung between 60 degrees and -60 degrees. Finally, we observed that the values of the torsional angles of the three linkages: NeuAc(alpha 2-6)Gal(beta 1-), Gal(beta 1-4)GlcNAc(beta 1-) and GlcNAc(beta 1-2)Man(beta 1-) of each of the two antennae were different, demonstrating their asymmetric conformation.     

Crystalline conformation of homo- and regular heteroglucan chains

Crystalline polysaccharides with 1 → 3−β and 1 → 4−β glycosidic linkages are the most prevalent ones in nature. An interpretation of recent X-ray data on 1 → 3−α glucan shows that it has a ribbon-like crystalline conformation similar to cellulose. Comparison of the crystalline conformation of the four principal homoglucans shows that they fall either in the ‘ribbon-like’ or ‘large amplitude’ helix class. Heteroglucans with a regular sequence of glucosidic linkages show characteristics of the ‘extended conformation’ rather than the ‘coiled conformation’ even when there is 50% of a linkage which in a homoglucan leads to a large amplitude helix. It is concluded that X-ray diffraction analysis fully establishes the hypothesis that the glycosidic linkage type is the determinant of polysaccharide conformation. In this respect, polysaccharides are more like synthetic polymers than proteins or nucleotides; in the latter, it is variation in the substituents which are responsible for the conformational diversity.     

Solution conformations of a trimannoside from nuclear magnetic resonance and molecular dynamics simulations

N-linked oligosaccharides often act as ligands for receptor proteins in a variety of cell recognition processes. Knowledge of the solution conformations, as well as protein-bound conformations, of these oligosaccharides is required to understand these important interactions. In this paper we present a model for the solution conformations sampled by a simple trimannoside, methyl 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, which contains two of the most commonly found glycosidic linkages in N-linked oligosaccharides. This model was derived from simulated annealing protocols incorporating distance restraints extracted from NOESY spectra along with torsional restraints computed from three-bond (1)H-(13)C coupling constants measured across the glycosidic bonds. The model was refined in light of unrestrained molecular dynamics simulations conducted in the presence of solvent water. The resulting model depicts a molecule undergoing conformational averaging in solution, adopting four major and two minor conformations. The four major conformations arise from a pair of two-state transitions, one each at the alpha(1-->3) and alpha(1-->6) linkages, whereas the minor conformations result from an additional transition of the alpha(1-->6) linkage. Our data also suggest that the alpha(1-->3) transition is fast and changes the molecular shape slightly, whereas the alpha(1-->6) is much slower and alters the molecular shape dramatically.     

Interactions of wheat-germ agglutinin with GlcNAc beta 1,6Gal sequence

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.     

A molecular mechanical force field for the conformational analysis of oligosaccharides: comparison of theoretical and crystal structures of Man alpha 1-3Man beta 1-4GlcNAc

A molecular mechanical force field is described for the conformational analysis of oligosaccharides. This force field has been derived by the addition of new parameters to the AMBER force field and is compatible with simulations of proteins. This new parametrization is assessed by comparison of the theoretically predicted conformations of Man alpha 1-3Man beta 1-4GlcNAc with the corresponding crystal structure. Molecular dynamics simulation data are presented for this structure both in vacuo and with the explicit inclusion of water molecules. While the former demonstrate significant torsional oscillations about glycosidic linkages at physiological temperature, in the latter these oscillations are highly damped due to the stabilizing influence of a "cage" of solvent-solvent and solvent-solute hydrogen bonds.