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Three-dimensional structures of oligosaccharides
structures of oligosaccharides
Three-dimensional
Robert University
Oligosaccharides
Woods
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Current Opinion
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5:591-598
conformations if those conformations balanced with regard to other energetic
Introduction Unlike oligopeptides, oligosaccharides cannot yet be characterized in terms of their three-dimensional or secondary structural motifs, although these motifs may exist. However, the inherent flexibility of many oligosaccharides in solution leads to structures that are frequently best described as ensembles of distinct conformers. Several structural and electronic contributions combine to achieve this effect [l]. Nowhere is the interplay between theory and experiment more necessary than in the conformational analysis of oligosaccharides. This review evaluates advances in both areas during the past year. The polyhydroxylated nature of sugars has long been recognized as facilitating the formation of extensive hydrogen-bonding networks in the solid state [2]. More recently, hydrogen-bonding networks have been predicted to exist in the gas phase [3-51 and to a lesser extent in aqueous sugar solutions [1,6]. Although inter-residue hydrogen bonding has been predicted to exist in solution in several systems [1,7], its existence may reflect in part the presence of a stable conformation, in which the hydroxyl groups are located fortuitously within hydrogen-bonding distance of one another. The energy of a water-water or sugar-water hydrogen bond is comparable to that of an interaction between carbohydrate hydroxyl groups, such that internal hydrogen bonding may yield little enthalpic benefit. However, the energetic stabilization afforded by hydrogen-bond cooperativity may be greater than that predicted from a simple summation of individual hydrogen-bond energies [8,9]. Further, hydrogen bonding might be expected to play a deciding role in selecting between two or more
were essentially contributions.
Steric and hydrophobic interactions may play significant roles in defining oligosaccharide conformation, as indicated by the frequency with which computational methods that completely ignore both solvation and hydrogen bonding can generate conformations that are consistent with solution NMR data [10-151. These methods assume that the conformation of an oligosaccharide is dependent only on van der Waals interactions and on the presence of an exe-anomeric effect [lb-181. This review has been divided into three major areas as a function of the primary method of analysis, namely NMR spectroscopic, X-ray crystallographic, and theoretical methods. Although recent interesting applications have involved other experimental techniques, such as fluorescence-labelling [19] and optical rotation [20*,21’], the majority of the reports for the past year can be coarsely divided into these three categories.
NMR-derived
conformations
The characteristic lack of inter-residue nuclear Overhauser effects (NOES) limits the ability of IH-NMR to establish uniquely the conformation of an oligosaccharide in solution [22]. Consequently, it is necessary to turn frequently to computational support in interpreting NMR data, as well as in predicting probable (or improbable) conformations. These computational aspects have been reviewed very recently [23”]. The emergence of
Abbreviations Fuc-fucose;
Gal-galactose; Me-methyl;
hCC-human
MM-molecular
chorionic gonadotropin; Man-mannose;
modeling;
Neu5Ac-Kacetylneuraminic
ROE-rotating-frame
0 Current
Overhauser
Biology
effect;
MC-Monte
acid; tr-NOE-transferred
Ltd ISSN 0959-440X
NOE-nuclear
Carlo; MD-molecular Overhauser
dynamics;
effect;
NOE.
591
592
Carbohydrates and glycoconjugates the ability to observe useful in augmenting
labile protons in water should prove available NOE data [7,24-261.
Most conformational studies employ one or more of the following computational methods: energy mapping, Monte Carlo (MC) sampling, or molecular dynamics simulation. Each method relies on one of (MD) several mathematical expressions (force fields) that relate carbohydrate conformation to energy. Experimental 13ClH J-coupling constants may be used to further define the conformations of glycosidic linkages. Although isotopic enrichment can be employed effectively to overcome the experimental obstacles that arise from the naturally low abundance of 1% [27*,28], general advances in the measurement of trans-glycosidic heteronuclear J-couplings [29**,30’], and 13ClH spectra [31**] have also been reported. Attempts to correlate 13C chemical shifts to glycosidic-linkage conformation may offer a supplementary source of conformational data [32]. Valuable information pertaining to the dynamics and molecular orientation may also be gained by anchoring the hydrophobic tails of lipid-linked carbohydrates to micelles or bilayers [33,34-l. Recent NMR developments in the carbohydrate field have been reviewed elsewhere [35**]. The NMR-based conformational analysis of oligosaccharides generally proceeds in one of two directions: either to determine the solution conformation for its own sake, or to establish the influence on that conformation from binding to a protein. In the first case, the traditional approach has been to employ distance-mapping procedures, often with the hard sphere ch-o-anomeric (HSEA) force field [12,14], to determine sterically allowed and NOE-consistent conformations. More recently, the application of MC and MD sinulations has become increasingly popular [1,36-391. A detailed example of the combination of an MC method with NOE data, applied to a disaccharide, has been reported by Weimar et al. [39]. The MD and MC methods are popular because they provide ensemble averages of conformations, which are often more representative of the properties of solvated oligosaccharides than would be any single conformation. In the analysis of the effect of binding on conformation, transferred NOE (tr-NOE) experiments may be invaluable for observing changes in the NOE intensities as a function of the bound conformation [40**]. A review of tr-NOE experiments has appeared recently [41”]. Interpretation of these intensity changes in terms of conformational changes requires a suitable molecular model; however, the flexibility of the oligosaccharide in the bound conformation may be so reduced that it is unnecessary to determine an ensemble of conformations. In their analysis of a blood group H trisaccharide, Widmalm and Venable [42*] performed MD simulations both in vacua and with explicit consideration of solvent with a CHARMm-type carbohydrate force field [43,44]. Surprisingly, the in vacua simulations indicated that
the use of either a constant dielectric of 1 D (pure vacuum), or a distance-dependent dielectric, leads to better reproduction of the NMR-derived inter-residue distances than does a dielectric of 80 D (water). Although a simulation with explicit solvation also gave good agreement [42-l, it was performed for only a very short duration (100 ps). The simulations indicated similar properties, both in terms of linkage flexibility for the L-Fuca(l,2)1>-Galp linkage (Fuc, fucose; Gal, galactose), and cooperativity of conformational changes associated with the @ and y glycosidic angles. In contrast, MD simulations on methyl a-lactoside [45*], employing a carbohydrate parameterization of the AMBER force field [46,47], led to the conclusions that a dielectric constant of ~40 D results in improved agreement with experimental data, but that in the absence of explicit solvent the data from the simulations should be treated qualitatively. In order to overcome many of these apparent discrepancies the use of rotating-frame Overhauser effect (ROE)or NOE-determined inter-proton distances as restraints on the simulation has been advocated [48]. Such restraints can bias the simulation or, worse, lead to artifactual or virtual conformations, as discussed elsewhere [49,50”]. When oligosaccharides exist primarily in a single conformation in solution, however, the use of NMR-derived inter-proton distance restraints can lead rapidly to the determination of an NMR-consistent conformation [51]. The Lewisa (Lea) and Lewisx (LeX) blood-group antigens and their derivatives continue to be the subjects of conformational studies. As classes, these molecules have been shown to exist in single conformations in solution. A single conformation does not necessarily imply that the oligosaccharide is rigid, merely that the glycosidic torsion angles exhibit only small oscillations around otherwise constant average values. Neither sialylation [38] nor sulfation [52] of the terminal galactose residue appears to alter the conformational properties of the Le antigenic core residues. Cooke et al. [53**] reported recently that the conformation of sialyl LeX alters upon binding to E-selectin, and proposed that the conformational change is restricted to the Neu5Aca(2,3)Gal (Neu5Ac, N-acetyl neuraminic acid) linkage. The conformational flexibility of the Neu5Aca(2,3)Gal linkage, also present in gangliosides, has been found to depend on the ganglioside sequence [33,54,55**]. Flexibility about this linkage has been predicted recently from NMR data alone [56] and in combination with MD simulations, both with [38] and without [57,58-l inter-proton distance restraints. Protein-induced conformational changes in a trisaccharide, related to the antigenic determinant of a have been observed in trSalrnorzella polysaccharide, NOE experiments [40**]. The tr-NOE-derived distance restraints were shown to be consistent with an X-ray structure of the same complex, but not with the solution conformation of the trisaccharide [40”]. Wyss
Three-dimensional structures of oligosaccharides Woods tt af. [59-l noted that the chemical shifts of only the N-acetylglucosamine core residues in high-mannose oligosaccharides were significantly altered by N-linkage to human protein CD2, perhaps indicating that protein-induced conformational changes may be highly localized. In their model for the complexation of polysialic acid with its monoclonal antibody, Evans et al. [60*] found that acceptable shape and charge complementarity could be achieved without altering the conformation of the polysaccharide f?om its proposed solution conformation [61]. It should be noted that interactions between oligosaccharides and proteins can also alter temporal properties, such as flexibility and overall dynamics [34*,62]. Linear oligosaccharides frequently exhibit no interresidue NOE contacts that extend beyond pairs of residues [63*,64]. Thus, it may be extremely difficult to determine with certainty the overall conformation of the carbohydrate chain. In highly heterogeneous sequences, a linear oligosaccharide may form a relatively compact conformation [64]; however, in many polysaccharides and homopolymers a helical conformation is implied [37,61,63*,65]. On the basis of energy minimization and MD simulations, Bernabk et al. [63’] have shown that the NMR data for tri- and tetrasaccharides of fi-glucose, with alternating 1,3 and 1,4 linkages, are consistent with extended overall conformations, in which each glycosidic torsion angle adopts a conformation also seen in the relevant disaccharides. Extended conformations were also predicted for homopolymers of arabinofuranose [66*]. In contrast, Liu ct al. [67] did not observe coalescence of the 1% signals in a2,1-linked tiuctofuranosyl polysaccharides, and concluded that simple helical conformations are not preponderant in solution. In that example it appeared that extrapolation of the conformations of related oligosaccharides [68] led to a polymer in which the resultant helix would be highly strained [67].
terminus of the tetrasaccharide appeared to occupy two conformational states. Staffer et al. [70*] suggest that this unusual result may have arisen from energetic contributions related to hydrogen bonding, or from partial ionization of an amino acid residue. In contrast, Zdanov et al. [71-l have reported that in the case of binding of a trisaccharide antigen to a single-chain antibody Fv fragment, a different conformation was found for the trisaccharide than had previously been reported for binding of the same trisaccharide to an antibody Fab fragment [40**]. A study of the effect of ionic oligosaccharide substituents on binding between a lectin and derivatives of Leb has been reported [72]. In studies of the bound conformations of a complex biantennary oligosaccharide [73] with a legume lectin and with galectin-1, Bourne et al. [74*-l concluded that the majority of the observed values of the $ and v angles all corresponded to low-energy regions of the potential energy surface, with the exception of those of the Mana(l,6)Man linkages, which they reported differed slightly from the absolute minimumenergy conformation. The energies for these unusual conformations (@160”) have been estimated to be -5 kcal/mol above the minimum-energy conformation, in which @ = 70” [75]. This observation appears to conflict with the conclusion of Bourne et al. that galectiri-1 selects oligosaccharide conformations from an ensemble of low-energy conformations present in solution [74**]. However, the accuracy of this energy estimate is uncertain. Similarly, in a study of the binding of maltotetraose to @amylase [76-l, the tetrasaccharide adopted a conformation in which some of the $I angles were distorted from values predicted for isolated molecules [77], but were similar to those found in related crystal structures [78].
Although evidence continues to emerge to support the view that the conformational properties of an oligosaccharide in solution may be reasonably approximated by those of the constituent disaccharides, the same cannot be said for oligosaccharides in protein complexes. Several crystal structures of oligosaccharide-protein complexes have been reported in which the crystalline environment induces conformational changes in the oligosaccharide [69,70’,71’].
To address the effects of binding on oligosaccharide conformation, Imberty and Pirez, and Loris et al. [79**,80*] have reported a molecular-docking procedure and a conformational-search procedure to orient the carbohydrate, and the preliminary results are encouraging. Methods that focus entirely on the isolated oligosaccharide may also be useful, at least to the extent that the bound and free conformations are similar. Qasba er al. [81] have reported that during MD simulations an isolated oligosaccharide may adopt conformations similar to that found in oligosaccharide-protein complexes, even if only very transiently. Moreover, in a study of legume lectin-glycopeptide binding, Bourne et al. [73] reported that the region of the oligosaccharide that was most intimately involved in binding to the protein was also most similar in conformation to the free oligosaccharide.
Two examples which illustrate that protein binding may alter the oligosaccharides’ conformation in regions either proximal or distal to the actual binding site have been reported [70’,71’]. In a study of the binding of the glucoamylase inhibitor I,-Xbro-dihydroacarbose to the enzyme, it was observed [70’] that the non-bound
Although the conformational properties of polysaccharides per se are not covered in this review, two X-ray fiber diffraction studies that illustrate the necessity of employing molecular modeling (MM) techniques in the analysis are worth mentioning. Yui et al. [82] have applied energy-mapping techniques with
X-ray crystallographic
studies
593
594
Carbohydratesand glycoconjugates the PFOS force field [83] to derive a left-handed helical structure for the acidic heteropolysaccharide glucurono-xylo-mannan, which is consistent with the crystallographically deternfined 24.6 A fiber repeat distance. This helix is stabilized by interactions between the acidic side-chain residues and the mannan backbone. A somewhat similar structural topology was also seen for an anionic galactoglucan, in which the acidic side-chain residues interact with the backbone [84°]. However, in this example, the helix is right-handed with a pitch o f 15.9 fi~. Both polysaccharide models claim to be consistent with low-energy conformations of the individual glycosidic linkages. Ill an attempt to further define the conformation of cellulose II, Gessler et al. [85"] reported the crystal structure o f ~-l)-cellotetraose. This oligosaccharide exhibited the same molecular packing as found for cellulose I1, suggesting that the hydroxymethyl groups in cellulose II may all adopt a similar conformation, in contrast to earlier models. A thorough review o f the conformational properties of hydroxymethyl groups has appeared [86].
Molecular modeling Advances continue to be made in carbohydrate-modeling methods, and these may ultimately facilitate accurate conformational analysis to complement experimental methods [87%88",89]. However, the uncertain accuracy of many current carbohydrate force fields generally necessitates that oligosaccharide modeling rely heavily on experimental data [48]. Fundamental differences between modeling methodologies are found in their approaches to the treatment of water, which vary from complete neglect [36,40",42",55",90], to a continuum model [37,38,47,50",52,55",63",64,66",90,91], to explicit inclusion o f individual solvent molecules [1,37,51,58"]. In MD simulations of high-mannose oligosaccharides, performed using the AMBER. Ibrce field in vacuo, Balaji et al. [91] have reported that the two or1,3 and ctl,6 linkages display different conformations. They concluded that it may not always be correct to assume that the conformational behavior of the linkages in an oligomer will be equivalent to that of the constituent disaccharides. However, Balaji et al. [91] reported only qualitative agreement with some NMP,, parameters. Moreover, given their recent report [81], which indicated that, in contrast to N M R data, this computational method predicted an equal population of all C5-C6 rotamers for the ctl,6 linkage, the high-mannose sinmlations [91] should be considered as identifying merely sterically possible conformations. Although sialic acids have frequently been the subject of theoretical investigations, the physiologically relevant counter ion has been ignored until recently. Mukhopad-
hyay and Bush [58"] have reported C H A R M m MD simulations of the sodimn salt of the disaccharide Neu5Acc~(2,3)Gal[~OMe (Me, methyl) in explicit water. In their constant volume simulations explicit water itltroduced a damping of the glycosidic oscillations relative to that found from simulations in vacuo. Notably, these authors reported that this linkage exists as an equilibrium among muhiple conformations, unlike earlier studies which suggested a more rigid conformation. in vacuo approach, involving constrained MD sinmlations, to discern the role o f the Na + counter ion in altering the conformation o f the sialic acid-containing pentasaccharide of ganglioside GM1, was reported by R`odgers and Portoghese [90]. The Na + was found to prefer to coordinate to a large number of oxygen atoms in molecular pockets, frequently including coordmation to the carboxylate group. Not surprisingly, this coordination was observed to lead to changes in conformational relative energies, compared with Na+-free simulations. 1Kodgers and Portoghese [90] observed that Na+-oligosaccharide dissociation occurs when the presence of water is approximated by a bulk dielectric constant, a feature which they claim would be hindered in the presence of explicit solvent molecules. Ion diffusion was not seen in the explicit solvent model of Mukhopadhyay and Bush [58"]. All
Two approaches to modeling the conformations of N-linked oligosaccharides in the presence of their protein cores were recently reported [92%93°]. Lapthorn et al. [92"] overlayed the crystallographic coordinates for the hydrogen fluoride-truncated N-acetylated chitobiose core, present in their X-ray structure of human chorionic gonadotropin (hCG), with the crystallographically deternfined coordinates of a complex biantennary oligosaccharide from thrombin. The generality of this approach is uncertain, as the truncated carbohydrate core may not typically adopt the same orientation relative to the protein surface as it would in the intact oligosaccharide. Woods et al. [93"] have modeled glycosylated bovine ribonuclease B by combining the X-ray data for the protein [94], in which the carbohydrate was intact but unresolved in the electron-density map, with a high-mannose N-linked oligosaccharide conformation predicted from MD simulations in the presence of explicit water. In the case o f ribonuclease B [93"], the conformation of the oligomannose is similar to that determined by NMR` for the same oligosaccharide N-linked to CD2 [59"].
Conclusions Considerable progress has been made in all aspects of oligosaccharide conformational analysis, and it is becoming increasingly possible to determine accurately the average solution conformation of an oligosaccharide, as well as its conformation in protein complexes.
Three-dimensional structures of oliaosaccharides Woods However, quantification of the intuitive concept of molecular flexibility, or rigidity, is essential before adequate comparisons of the relative dynamic properties of oligosaccharides can be achieved. X-ray and tr-NOE data from carbohydrate-protein complexes provide valuable information regarding the bound conformation of the carbohydrate, and indicate that proteins may induce confornlational changes in the oligosaccharide. On the basis of the current data it seems unlikely that in all cases the protein selects oligosaccharide conformations that pre-exist in solution. A complete understanding of binding phenonlena necessitates detailed study of the role of water in influencing oligosaccharide conformation. Moreover, the fundamental properties (structural, and energetic) associated with oligosaccharide-oligosaccharide and oligosaccharide-protein interactions require careful continuing assessment.
References and recommended Papers of particular interest, published review, have been highlighted as: - . of special interest .. of outstanding interest
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40. ..
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Klein RA, Hartmann R, Egge H, Behr T, Fischer W: The aqueous solution structure of the tetrasaccharide-ribitol repeat-unit from the lipoteichoic acid of Streptococcus pneumoniae strain R6 determined using a combination of NMR spectroscopy and computer calculations. Carbohydr Res 1994, 256: 189-222.
38.
Rutherford TJ, Spackman DC, Simpson PJ, Homans SW: 5 nanosecond molecular dynamics and NMR study of conformational transitions in the sialyl-Lewis X antigen. c/ycofJio/ogy 1994, 4:59-68.
39.
Weimar T, Meyer 8, Peters T: Conformational analysis of aD-Fuc-(l-4)-p-D-ClcNAc-OMe. One-dimensional transient NOE experiments and metropolis Monte Carlo simulations. / Biomol NMR 1993, 3:399-414.
and solution conformations 1987, 26:6664-6676.
50. ..
Rutherford TJ, Homans SW: Restrained vs free dynamics simulations of oligosaccharides: application to solution dynamics of biantennary and bisected N-linked glycans. Biochemisfry 1994, 33:9606-9614. MD simulations performed for 510 ps in vacua both with and without NOE-derived distance restraints indicate that the conformatlons oi bisected glycans are consistent with previously predicted rigid structures. 51.
Homans S, Forster M: Application of restrained minimization, simulated annealing and molecular dynamics simulations for the conformational analysis of oligosaccharides. Clycobiology 1992, 2:143-l 51.
52.
Kogelberg H, Rutherford TJ: Studies on the three-dimensional behavior of the selectin ligands Lewis’ and sulphated Lewisa using NMR spectroscopy and molecular dynamics simulations. C/ycobio/ogy 1994, 4:49-57.
53. ..
Cooke RM, Hale RS, Lister SC, Shah G, Weir MP: The conformation of the sialyl Lewis X ligand changes upon binding to E-seleclin. Biochemistry 1994, 33:10591-l 0596. Tr-NOE studies demonstrate a conformational change in the oligosaccharide upon binding to the selectin. The bound conformation may also exist
Three-dimensional structures of olkosaccharides Woods Cros 5, lmberty A, Bouchemal N, tie& du Penhoat C, P&ez S: Modeling of arabinofuranose and arabinan. II. NMR and conformational analysis of arabinobiose and arabinan. Biopolymers 1994, 34:1433-l 447. No single glycosidic conformation is found that is consistent with the NMR data; rather, an ensemble of several low-energy conformations is predicted. Extrapolation to models for the polysaccharide leads to several possrble helical structures.
to a limited extent in solution; however, other solution conformations must be present lo explain the observed NOE data.
66. .
Acquotti D, Poppe L, Dabrowski J, van der Lierh CW, Sonnino S, Tettamanti C: Three-dimensional structure of the oligosaccharide chain of CM1 ganglioside revealed by a distance-mapping procedure: a rotating and laboratory frame nuclear Overhauser enhancement investigation of native glycolipid in dimethyl sulfoxide and in water-dodecylphosphocholine solutions. f Am Cbem Sot 1990, 112:7772-7778.
67.
Liu J, Waterhouse AL, Chatterton NJ: Do inulin oligomers adopt a regular helical form in solution? I Carbohydr Chem 1994, 13:859-872.
68.
French AD, Mouhous-Riou N, Perez S: Computer modelling of the tetrasaccharide nystose. Carbohydr Res 1993, 247:51-62.
69.
Merritt EA, Sarfaty S, Van der Akker F, L’Hoir C, Martial ]A, HOI WCJ: Crystal structure of cholera toxin B-pentamer bound to receptor G,,,, pentasaccharide. Proleirl Sri 1994, 3:16&l 75.
54.
Acquotti D, Cantu L, Ragg E, Sonnino 5: Geometrical of ganglioside CalNAcand conformational properties ELM J Cola, IV%alNAcIV3-Neu5Acll3NeuSAcGgOse.&er. Biochem 1994, 225:271-288. Laser light-scattering experiments indicate that micellar CalNAc-CD,, exhibits a molecular mass 22% larger than that for mlcellar GD]a. This unexpectedly large difference is explained on ihe basis of conformational and dynamic properties determined by restrained MM and MD simulations. 55. ..
56.
Wormald MR, Edge CJ: The systematic use of negative nuclear Overhauser constraints in the determination of oligosaccharide conformations: application to sialyl-Lewis X. Carbohydr Res 1993, 246:337-344.
57.
Mukhopadhyay oligosaccharide 34:21-29.
C, Miller receptor
KE, Bush CA: Conformation for E-selectin. Kopolymers
of the 1994,
Mukhopadhyay C, Bush AC: Molecular dynamics simulation of oligosaccharides containing Kacetyl neuraminic acid. Biopolymers 1994, 34: 11-20. Reports a fully solvated simulation (including counter ion) for this challenging system. A good summary of the problems associated with NOE data interpretation is also presented.
58. .
Wyss DF, Choi JS, Wagner C: Composition and sequence specific resonance assignments of the heterogeneous N-linked glycan in the 13.6 kDa adhesion domain of human CD2 as determined by NMR on the intact glycoprotein. Biochemisrry 1995, 34:1622-l 634. Extensive, although preliminary, NMR study of an intact glycoprotein which had as its aim derivation of the overall three-dimensional structure. A typographical error in [93*] led Wyss ef al. to conclude incorrectly that their conformation was not in good agreement with that proposed in [93-J. 59. .
Evans SV, Sigurskjold SW, Jennings HJ, Brisson J-R, To R, Tse WC, Altman E, Frosch M, Weisgerber C, Kratzin HD, et al.: Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for a-(2-8)-polysialic acid. Bjochemistry 1995, 34:6737-6744. A comprehensive study which combines X-ray, thermodynamic and modeling dara to produce a consistent model for the binding of a polysaccharide 161 I with an IgG binding fragment. 60. .
61.
Brisson J-R, Baumann H, lmberty A, Perez S, Jennings HJ: Helical epitope of the group 6 meningococcal a(2-8)-linked sialic acid polysaccharide. Biochemisrry 1992, 31:4996-5004.
62.
Joao HC, Scragg IC, Dwek RA: Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B. FE65 Lelt 1992, 307:343-346.
63. .
Berna& M, Jim&ez-Barber0 J, Planas A: The conformation of the tri- and tetrasaccharide produced in the hydrolysis of barley glucan with the enzyme endo-1,3-1,4+glucan 4-glucanohydrolase from f?aci/lus licheniformis. / Carbohydr Chem 1994, 13:799-817. Unrestrained MD simulations in vacua and NOE data lead to the conclusion that the glycosidic linkages in the oligosaccharide adopt low-energy conformations. 64.
65.
Weller CT, McConville M, Homans SW: Solution structure and dynamics of a glycoinositol phospholipid (GIPL-6) from Leishmania major. Biopolymers 1994, 34:1155-l 163. Tezuka Y: Nuclear Overhauser effect spectroscopy (NOESY) detection of the specific interaction between substituents in cellulose and amylose triacetates. Biopolymers 1994, 34:i 477-1482.
70. .
Staffer B, Aleshin AE, Firsov LM, Svensson B, Honzatko RB: Refined structure for the complex of o-gluc*dihydroacarbose with glucoamylase from Aspergihs awamori var. Xl00 lo 2.2 A resolution: dual conformations for extended inhibitors bound to the active site of glucoamylase. FEBS Lerf 1995, 358:57-61. Two of the sugar residues that interact directly with the protein are well resolved; however, an optimal fit to the electron density for the remaining two residues is achieved only when two conformations are considered with a partial occupancy ratio of 35:65. 71. .
Zdanov A, Li Y, Bundle DR, Deng S-J, MacKenzie CR, Narang SA. Young NM, Cygler M: Structure of a single-chain antibody variable domain_ fragment complexed with a carbohydrate antigen at 1.7-A resolution. Proc Nat/ Acad Sci USA 1994, 91~6423-6427. The positions of water molecules in the vicinity of the antigen, and their influence on the resulttng hydrogen-bond networks, are shown to alter the conformational preferences of the antrgen. 72.
Lemieux RU, Du M-H, Spohr U: Relative effect of ionic and neutral substituents on the binding of an oligosaccharide by a protein. / Am Chem Sot 1994, 116:9803-9804.
73.
Bourne Y, Mazurier I, Legrand D, Rough P, Montreuil J, Spik C, Cambillau C: Structures of a legume lectin complexed with the human lactotransferrin N2 fragment, and with an isolated biantennary glycopeptide: role of the fucose moiety. Slructure 1994, 2:209-219.
74. ..
Bourne Y, Bolgiano B, Liao D-I, Srrecker C, Cantau P, Herzberg 0, Feizi T, Cambillau C: Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides. Nature Strucr Bio/ 1994, 1:863-870. Well-resolved X-ray structures reveal unusual conformations for the Mana(l,6)Man linkages, arising not from flexibrlity associated with Ihe w angle, but rather from unusual @ angles. 75.
lmberty A, Gerber S, Tran V, Perez 5: Data bank of three-dimensional structures of disaccharides, a tool to build 3-D structures of oligosaccharides. Glycoconjugate / 1990, 7~27-54.
76. .
Mikami 8, Degano M, Hehre EJ, Sacchettini JC: Crystal structures of soybean B-amylase reacted with J%maltose and maltak active site components and their apparent roles in catalysis. Biochemistry 1994, 33:7779-7787. The maltal structure illustrates the delicate balance between conformationally conflicting interactions, namely between inter-residue hydrogen bonding and the exe-anomeric effect. 77.
Brady JW, Schmidt RK: The role of hydrogen bonding in carbohydrates: Molecular dynamics simulations of maltose in aqueous solution. / Phys Chem 1993, 97:958-966.
78.
Goldsmith E, Fletterick RJ: Oligosaccharide conformation and protein saccharide interactions in solution. Pure Appl Chem 19133, 551577-588.
79. ..
lmberty A, Perez S: Molecular modelling of proteinxarbohydratc interactions. Understanding the specificities of two legume lectins towards oligosaccharides. Clycobiology 1994, 4:351-366. Valuable advances in methods for molecular docking and conformational analysis of bound oligosaccharides are Introduced.
597
598
Carbohydrates and glycoconjugates 80. .
extensive revisions to the non-bonded and torsional terms of the AMBER force field.
81.
Clennon TM, Zheng Y-J, LeGrand SM, Shut&erg BA, KM Jr.: A force field for monosaccharides and (l-4) polysaccharides. / Compur Chem 1994, 15:1019-l 040. A parameter set relevant to the titled compounds is introduced, on improved treatment of non-bonded interactions in the AMBER field.
Loris R, Casset F, Bouckaert J, Pletinckx J, Dao-Thi M-H, Poortmans F, lmberty A, Perez S, Wyns L: The monosaccharide binding site of lentil lectin: an X-ray and molecular modelling study. Clycoconjugate ) 1994, 11:507-517. An application of a molecular docking protocol [76*1 is presented, together with an analysis of the energetics associated with carbohydrate binding. Qasba PK, Balaji PV, Rao VSR: Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle psi for the orientation of a 1,6-arm. Glycobiology 1994, 4:805-815.
82.
Yui T, Ogawa K, Kakuta M, Misaki A: Chain conformation of a glucurono-xylo-mannan isolated from fruit body of Tremelfa fuciformis berk. 1 Carbohydr Chem 1995, 14:255-263.
83.
Tvaroska I, Perez S: Conformational-energy calculations for oligosaccharides: a comparison of methods and a strategy of calculation. Carbohydr Res 1986, 149:389410.
Chandrasekaran R, Lee EJ, Thailambal VC, Zevenhuizen LPTM: Molecular architecture of a galactoglucan from Rhizobium Meliloti. Carbohydr Res 1994, 261:279-295. Interestingly, in this galactoglucan the interaction between the carboxylate group and the backbone appears to be modulated by bridging water molecules.
88.
.
Merz linked based force
89.
Howard AE, Cieplak P, Kollman PA: A Molecular mechanical model that reproduces the relative energies for chair and twistboat conformations of 1,3-dioxanes. / Comput Chem 1995, 16:243-261.
90.
Rodgers JC, Portoghese PS: Molecular modeling of the conformational and sodium ion binding properties of the oligosaccharide component of ganglioside CM1 . Biopolymers 1994, 34:1311-1326.
91.
Ealaji PV, Qasba PK, Rao VSR: Molecular dynamics simulations of high-mannose oligosaccharides. Clycobiology 1994, 4:497-515.
84.
.
92. .
Cessler K, Krauss N, Steiner T, Betzel C, Sandmann C, Saenger W: Crystal structure of p-D-CdOtetraOSe hemihydrate with implications for the structure of cellulose II. Science 1994, 266:1027-l 029. In an interesting application of MD in the solid phase, micro-crystals of cellulose It are simulated under periodic boundary conditions and were shown to be consistent with proposed conformational changes.
93. Woods R], Edge CJ, Dwek RA: Protein surface oligosaccharides . and protein function. Nafure Sfruct Bio/ 1994, 1:499-501. A model for the presentation of the oligosaccharide relative to the protein is presented, based on fully solvated MD data for the isolated oligosaccharide. Note that ‘created van der Waals’ should read ‘created no significant van der Waals’ and ‘residue B’ should read ‘residue A’ on pages 500 and 501 respectively.
85. .
86.
Bock K, Duus J: A Conformational study of hydroxymethyl groups in carbohydrates investigated by tH NMR spectroscopy. / Carbohydr Chem 1994, 13:513-543.
Woods RJ, Dwek RA, Edge Cl, Fraser-Reid 6: Molecular mechanical and molecular dynamical simulations of glycoproteins and oligosaccharides. 1. GLYCAM 93 parameter development. / fhys Chem 1995, 99:3832-3846. A parameter set that is generally applicable to oligosaccharides is reported, based on ab inirio molecular orbital calculations and involving
Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, lsaacs NW: Crystal structure of human chorionic gonadotropin. Nature 1994, 369:455-461. A strategy for exploring possible spatial relationships between the carbohydrate and the protein is presented.
94.
Williams RL, Greene SM, McPherson A: The crystal structure of ribonuclease B at 2.5-A resolution. / Viol Chem 1987, 262:16020-16031.
87. .
l
Carbohydrate 220 Riverbend
Research Center, The Road, Athens, Georgia
Three-dimensional
Robert University
Oligosaccharides
Woods
J
of Georgia,
represent a particularly
Athens,
challenging
USA
class of molecules for
conformational
analysis.
methods
have
begun
behavior;
however, general rules governing their conformational
have
not
insights
yet
Recent advances in experimental to
emerged.
yield X-ray
into protein-bound
necessarily
represent
an oligosaccharide’s interactions
further and
insight
NMR
oligosaccharide
highly
populated
inherent
flexibility
into
techniques
in Structural
Biology
preferences provide
conformations.
1995,
vital
but these do not
and lack of strong
places extreme demands on theoretical
Current Opinion
conformational
may
conformations,
solution
and theoretical
their
Moreover,
intermolecular
methods.
5:591-598
conformations if those conformations balanced with regard to other energetic
Introduction Unlike oligopeptides, oligosaccharides cannot yet be characterized in terms of their three-dimensional or secondary structural motifs, although these motifs may exist. However, the inherent flexibility of many oligosaccharides in solution leads to structures that are frequently best described as ensembles of distinct conformers. Several structural and electronic contributions combine to achieve this effect [l]. Nowhere is the interplay between theory and experiment more necessary than in the conformational analysis of oligosaccharides. This review evaluates advances in both areas during the past year. The polyhydroxylated nature of sugars has long been recognized as facilitating the formation of extensive hydrogen-bonding networks in the solid state [2]. More recently, hydrogen-bonding networks have been predicted to exist in the gas phase [3-51 and to a lesser extent in aqueous sugar solutions [1,6]. Although inter-residue hydrogen bonding has been predicted to exist in solution in several systems [1,7], its existence may reflect in part the presence of a stable conformation, in which the hydroxyl groups are located fortuitously within hydrogen-bonding distance of one another. The energy of a water-water or sugar-water hydrogen bond is comparable to that of an interaction between carbohydrate hydroxyl groups, such that internal hydrogen bonding may yield little enthalpic benefit. However, the energetic stabilization afforded by hydrogen-bond cooperativity may be greater than that predicted from a simple summation of individual hydrogen-bond energies [8,9]. Further, hydrogen bonding might be expected to play a deciding role in selecting between two or more
were essentially contributions.
Steric and hydrophobic interactions may play significant roles in defining oligosaccharide conformation, as indicated by the frequency with which computational methods that completely ignore both solvation and hydrogen bonding can generate conformations that are consistent with solution NMR data [10-151. These methods assume that the conformation of an oligosaccharide is dependent only on van der Waals interactions and on the presence of an exe-anomeric effect [lb-181. This review has been divided into three major areas as a function of the primary method of analysis, namely NMR spectroscopic, X-ray crystallographic, and theoretical methods. Although recent interesting applications have involved other experimental techniques, such as fluorescence-labelling [19] and optical rotation [20*,21’], the majority of the reports for the past year can be coarsely divided into these three categories.
NMR-derived
conformations
The characteristic lack of inter-residue nuclear Overhauser effects (NOES) limits the ability of IH-NMR to establish uniquely the conformation of an oligosaccharide in solution [22]. Consequently, it is necessary to turn frequently to computational support in interpreting NMR data, as well as in predicting probable (or improbable) conformations. These computational aspects have been reviewed very recently [23”]. The emergence of
Abbreviations Fuc-fucose;
Gal-galactose; Me-methyl;
hCC-human
MM-molecular
chorionic gonadotropin; Man-mannose;
modeling;
Neu5Ac-Kacetylneuraminic
ROE-rotating-frame
0 Current
Overhauser
Biology
effect;
MC-Monte
acid; tr-NOE-transferred
Ltd ISSN 0959-440X
NOE-nuclear
Carlo; MD-molecular Overhauser
dynamics;
effect;
NOE.
591
592
Carbohydrates and glycoconjugates the ability to observe useful in augmenting
labile protons in water should prove available NOE data [7,24-261.
Most conformational studies employ one or more of the following computational methods: energy mapping, Monte Carlo (MC) sampling, or molecular dynamics simulation. Each method relies on one of (MD) several mathematical expressions (force fields) that relate carbohydrate conformation to energy. Experimental 13ClH J-coupling constants may be used to further define the conformations of glycosidic linkages. Although isotopic enrichment can be employed effectively to overcome the experimental obstacles that arise from the naturally low abundance of 1% [27*,28], general advances in the measurement of trans-glycosidic heteronuclear J-couplings [29**,30’], and 13ClH spectra [31**] have also been reported. Attempts to correlate 13C chemical shifts to glycosidic-linkage conformation may offer a supplementary source of conformational data [32]. Valuable information pertaining to the dynamics and molecular orientation may also be gained by anchoring the hydrophobic tails of lipid-linked carbohydrates to micelles or bilayers [33,34-l. Recent NMR developments in the carbohydrate field have been reviewed elsewhere [35**]. The NMR-based conformational analysis of oligosaccharides generally proceeds in one of two directions: either to determine the solution conformation for its own sake, or to establish the influence on that conformation from binding to a protein. In the first case, the traditional approach has been to employ distance-mapping procedures, often with the hard sphere ch-o-anomeric (HSEA) force field [12,14], to determine sterically allowed and NOE-consistent conformations. More recently, the application of MC and MD sinulations has become increasingly popular [1,36-391. A detailed example of the combination of an MC method with NOE data, applied to a disaccharide, has been reported by Weimar et al. [39]. The MD and MC methods are popular because they provide ensemble averages of conformations, which are often more representative of the properties of solvated oligosaccharides than would be any single conformation. In the analysis of the effect of binding on conformation, transferred NOE (tr-NOE) experiments may be invaluable for observing changes in the NOE intensities as a function of the bound conformation [40**]. A review of tr-NOE experiments has appeared recently [41”]. Interpretation of these intensity changes in terms of conformational changes requires a suitable molecular model; however, the flexibility of the oligosaccharide in the bound conformation may be so reduced that it is unnecessary to determine an ensemble of conformations. In their analysis of a blood group H trisaccharide, Widmalm and Venable [42*] performed MD simulations both in vacua and with explicit consideration of solvent with a CHARMm-type carbohydrate force field [43,44]. Surprisingly, the in vacua simulations indicated that
the use of either a constant dielectric of 1 D (pure vacuum), or a distance-dependent dielectric, leads to better reproduction of the NMR-derived inter-residue distances than does a dielectric of 80 D (water). Although a simulation with explicit solvation also gave good agreement [42-l, it was performed for only a very short duration (100 ps). The simulations indicated similar properties, both in terms of linkage flexibility for the L-Fuca(l,2)1>-Galp linkage (Fuc, fucose; Gal, galactose), and cooperativity of conformational changes associated with the @ and y glycosidic angles. In contrast, MD simulations on methyl a-lactoside [45*], employing a carbohydrate parameterization of the AMBER force field [46,47], led to the conclusions that a dielectric constant of ~40 D results in improved agreement with experimental data, but that in the absence of explicit solvent the data from the simulations should be treated qualitatively. In order to overcome many of these apparent discrepancies the use of rotating-frame Overhauser effect (ROE)or NOE-determined inter-proton distances as restraints on the simulation has been advocated [48]. Such restraints can bias the simulation or, worse, lead to artifactual or virtual conformations, as discussed elsewhere [49,50”]. When oligosaccharides exist primarily in a single conformation in solution, however, the use of NMR-derived inter-proton distance restraints can lead rapidly to the determination of an NMR-consistent conformation [51]. The Lewisa (Lea) and Lewisx (LeX) blood-group antigens and their derivatives continue to be the subjects of conformational studies. As classes, these molecules have been shown to exist in single conformations in solution. A single conformation does not necessarily imply that the oligosaccharide is rigid, merely that the glycosidic torsion angles exhibit only small oscillations around otherwise constant average values. Neither sialylation [38] nor sulfation [52] of the terminal galactose residue appears to alter the conformational properties of the Le antigenic core residues. Cooke et al. [53**] reported recently that the conformation of sialyl LeX alters upon binding to E-selectin, and proposed that the conformational change is restricted to the Neu5Aca(2,3)Gal (Neu5Ac, N-acetyl neuraminic acid) linkage. The conformational flexibility of the Neu5Aca(2,3)Gal linkage, also present in gangliosides, has been found to depend on the ganglioside sequence [33,54,55**]. Flexibility about this linkage has been predicted recently from NMR data alone [56] and in combination with MD simulations, both with [38] and without [57,58-l inter-proton distance restraints. Protein-induced conformational changes in a trisaccharide, related to the antigenic determinant of a have been observed in trSalrnorzella polysaccharide, NOE experiments [40**]. The tr-NOE-derived distance restraints were shown to be consistent with an X-ray structure of the same complex, but not with the solution conformation of the trisaccharide [40”]. Wyss
Three-dimensional structures of oligosaccharides Woods tt af. [59-l noted that the chemical shifts of only the N-acetylglucosamine core residues in high-mannose oligosaccharides were significantly altered by N-linkage to human protein CD2, perhaps indicating that protein-induced conformational changes may be highly localized. In their model for the complexation of polysialic acid with its monoclonal antibody, Evans et al. [60*] found that acceptable shape and charge complementarity could be achieved without altering the conformation of the polysaccharide f?om its proposed solution conformation [61]. It should be noted that interactions between oligosaccharides and proteins can also alter temporal properties, such as flexibility and overall dynamics [34*,62]. Linear oligosaccharides frequently exhibit no interresidue NOE contacts that extend beyond pairs of residues [63*,64]. Thus, it may be extremely difficult to determine with certainty the overall conformation of the carbohydrate chain. In highly heterogeneous sequences, a linear oligosaccharide may form a relatively compact conformation [64]; however, in many polysaccharides and homopolymers a helical conformation is implied [37,61,63*,65]. On the basis of energy minimization and MD simulations, Bernabk et al. [63’] have shown that the NMR data for tri- and tetrasaccharides of fi-glucose, with alternating 1,3 and 1,4 linkages, are consistent with extended overall conformations, in which each glycosidic torsion angle adopts a conformation also seen in the relevant disaccharides. Extended conformations were also predicted for homopolymers of arabinofuranose [66*]. In contrast, Liu ct al. [67] did not observe coalescence of the 1% signals in a2,1-linked tiuctofuranosyl polysaccharides, and concluded that simple helical conformations are not preponderant in solution. In that example it appeared that extrapolation of the conformations of related oligosaccharides [68] led to a polymer in which the resultant helix would be highly strained [67].
terminus of the tetrasaccharide appeared to occupy two conformational states. Staffer et al. [70*] suggest that this unusual result may have arisen from energetic contributions related to hydrogen bonding, or from partial ionization of an amino acid residue. In contrast, Zdanov et al. [71-l have reported that in the case of binding of a trisaccharide antigen to a single-chain antibody Fv fragment, a different conformation was found for the trisaccharide than had previously been reported for binding of the same trisaccharide to an antibody Fab fragment [40**]. A study of the effect of ionic oligosaccharide substituents on binding between a lectin and derivatives of Leb has been reported [72]. In studies of the bound conformations of a complex biantennary oligosaccharide [73] with a legume lectin and with galectin-1, Bourne et al. [74*-l concluded that the majority of the observed values of the $ and v angles all corresponded to low-energy regions of the potential energy surface, with the exception of those of the Mana(l,6)Man linkages, which they reported differed slightly from the absolute minimumenergy conformation. The energies for these unusual conformations (@160”) have been estimated to be -5 kcal/mol above the minimum-energy conformation, in which @ = 70” [75]. This observation appears to conflict with the conclusion of Bourne et al. that galectiri-1 selects oligosaccharide conformations from an ensemble of low-energy conformations present in solution [74**]. However, the accuracy of this energy estimate is uncertain. Similarly, in a study of the binding of maltotetraose to @amylase [76-l, the tetrasaccharide adopted a conformation in which some of the $I angles were distorted from values predicted for isolated molecules [77], but were similar to those found in related crystal structures [78].
Although evidence continues to emerge to support the view that the conformational properties of an oligosaccharide in solution may be reasonably approximated by those of the constituent disaccharides, the same cannot be said for oligosaccharides in protein complexes. Several crystal structures of oligosaccharide-protein complexes have been reported in which the crystalline environment induces conformational changes in the oligosaccharide [69,70’,71’].
To address the effects of binding on oligosaccharide conformation, Imberty and Pirez, and Loris et al. [79**,80*] have reported a molecular-docking procedure and a conformational-search procedure to orient the carbohydrate, and the preliminary results are encouraging. Methods that focus entirely on the isolated oligosaccharide may also be useful, at least to the extent that the bound and free conformations are similar. Qasba er al. [81] have reported that during MD simulations an isolated oligosaccharide may adopt conformations similar to that found in oligosaccharide-protein complexes, even if only very transiently. Moreover, in a study of legume lectin-glycopeptide binding, Bourne et al. [73] reported that the region of the oligosaccharide that was most intimately involved in binding to the protein was also most similar in conformation to the free oligosaccharide.
Two examples which illustrate that protein binding may alter the oligosaccharides’ conformation in regions either proximal or distal to the actual binding site have been reported [70’,71’]. In a study of the binding of the glucoamylase inhibitor I,-Xbro-dihydroacarbose to the enzyme, it was observed [70’] that the non-bound
Although the conformational properties of polysaccharides per se are not covered in this review, two X-ray fiber diffraction studies that illustrate the necessity of employing molecular modeling (MM) techniques in the analysis are worth mentioning. Yui et al. [82] have applied energy-mapping techniques with
X-ray crystallographic
studies
593
594
Carbohydratesand glycoconjugates the PFOS force field [83] to derive a left-handed helical structure for the acidic heteropolysaccharide glucurono-xylo-mannan, which is consistent with the crystallographically deternfined 24.6 A fiber repeat distance. This helix is stabilized by interactions between the acidic side-chain residues and the mannan backbone. A somewhat similar structural topology was also seen for an anionic galactoglucan, in which the acidic side-chain residues interact with the backbone [84°]. However, in this example, the helix is right-handed with a pitch o f 15.9 fi~. Both polysaccharide models claim to be consistent with low-energy conformations of the individual glycosidic linkages. Ill an attempt to further define the conformation of cellulose II, Gessler et al. [85"] reported the crystal structure o f ~-l)-cellotetraose. This oligosaccharide exhibited the same molecular packing as found for cellulose I1, suggesting that the hydroxymethyl groups in cellulose II may all adopt a similar conformation, in contrast to earlier models. A thorough review o f the conformational properties of hydroxymethyl groups has appeared [86].
Molecular modeling Advances continue to be made in carbohydrate-modeling methods, and these may ultimately facilitate accurate conformational analysis to complement experimental methods [87%88",89]. However, the uncertain accuracy of many current carbohydrate force fields generally necessitates that oligosaccharide modeling rely heavily on experimental data [48]. Fundamental differences between modeling methodologies are found in their approaches to the treatment of water, which vary from complete neglect [36,40",42",55",90], to a continuum model [37,38,47,50",52,55",63",64,66",90,91], to explicit inclusion o f individual solvent molecules [1,37,51,58"]. In MD simulations of high-mannose oligosaccharides, performed using the AMBER. Ibrce field in vacuo, Balaji et al. [91] have reported that the two or1,3 and ctl,6 linkages display different conformations. They concluded that it may not always be correct to assume that the conformational behavior of the linkages in an oligomer will be equivalent to that of the constituent disaccharides. However, Balaji et al. [91] reported only qualitative agreement with some NMP,, parameters. Moreover, given their recent report [81], which indicated that, in contrast to N M R data, this computational method predicted an equal population of all C5-C6 rotamers for the ctl,6 linkage, the high-mannose sinmlations [91] should be considered as identifying merely sterically possible conformations. Although sialic acids have frequently been the subject of theoretical investigations, the physiologically relevant counter ion has been ignored until recently. Mukhopad-
hyay and Bush [58"] have reported C H A R M m MD simulations of the sodimn salt of the disaccharide Neu5Acc~(2,3)Gal[~OMe (Me, methyl) in explicit water. In their constant volume simulations explicit water itltroduced a damping of the glycosidic oscillations relative to that found from simulations in vacuo. Notably, these authors reported that this linkage exists as an equilibrium among muhiple conformations, unlike earlier studies which suggested a more rigid conformation. in vacuo approach, involving constrained MD sinmlations, to discern the role o f the Na + counter ion in altering the conformation o f the sialic acid-containing pentasaccharide of ganglioside GM1, was reported by R`odgers and Portoghese [90]. The Na + was found to prefer to coordinate to a large number of oxygen atoms in molecular pockets, frequently including coordmation to the carboxylate group. Not surprisingly, this coordination was observed to lead to changes in conformational relative energies, compared with Na+-free simulations. 1Kodgers and Portoghese [90] observed that Na+-oligosaccharide dissociation occurs when the presence of water is approximated by a bulk dielectric constant, a feature which they claim would be hindered in the presence of explicit solvent molecules. Ion diffusion was not seen in the explicit solvent model of Mukhopadhyay and Bush [58"]. All
Two approaches to modeling the conformations of N-linked oligosaccharides in the presence of their protein cores were recently reported [92%93°]. Lapthorn et al. [92"] overlayed the crystallographic coordinates for the hydrogen fluoride-truncated N-acetylated chitobiose core, present in their X-ray structure of human chorionic gonadotropin (hCG), with the crystallographically deternfined coordinates of a complex biantennary oligosaccharide from thrombin. The generality of this approach is uncertain, as the truncated carbohydrate core may not typically adopt the same orientation relative to the protein surface as it would in the intact oligosaccharide. Woods et al. [93"] have modeled glycosylated bovine ribonuclease B by combining the X-ray data for the protein [94], in which the carbohydrate was intact but unresolved in the electron-density map, with a high-mannose N-linked oligosaccharide conformation predicted from MD simulations in the presence of explicit water. In the case o f ribonuclease B [93"], the conformation of the oligomannose is similar to that determined by NMR` for the same oligosaccharide N-linked to CD2 [59"].
Conclusions Considerable progress has been made in all aspects of oligosaccharide conformational analysis, and it is becoming increasingly possible to determine accurately the average solution conformation of an oligosaccharide, as well as its conformation in protein complexes.
Three-dimensional structures of oliaosaccharides Woods However, quantification of the intuitive concept of molecular flexibility, or rigidity, is essential before adequate comparisons of the relative dynamic properties of oligosaccharides can be achieved. X-ray and tr-NOE data from carbohydrate-protein complexes provide valuable information regarding the bound conformation of the carbohydrate, and indicate that proteins may induce confornlational changes in the oligosaccharide. On the basis of the current data it seems unlikely that in all cases the protein selects oligosaccharide conformations that pre-exist in solution. A complete understanding of binding phenonlena necessitates detailed study of the role of water in influencing oligosaccharide conformation. Moreover, the fundamental properties (structural, and energetic) associated with oligosaccharide-oligosaccharide and oligosaccharide-protein interactions require careful continuing assessment.
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