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

P.-G. Nyholm1,2, L.A. Mulard3, C.E. Miller4, T. Lew5, R. Olin2 and C.P.J. Glaudemans4

2Department of Medical Biochemistry and Centre for Structural Biology, University of Göteborg, Medicinaregatan 7, 405 30 Göteborg, Sweden; 3Unité de Chimie Organique, Institut Pasteur, 28 rue de Dr. Roux, 75724 Paris Cedex 15, France; 4Section on Carbohydrates, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA; and 5Department of Medical Genetics, University of Toronto, Toronto, Ontario M5S1A8, Canada

Received on March 23, 2001; revised on June 11, 2001; accepted on June 27, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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} {approx} –40°/–30° (I) and {approx} 15°/30° (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.

Key words: conformation/molecular mechanics/molecular modeling/O-antigen/Shigella


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Shigella dysenteriae type 1 (Shiga, 1898Go) gives rise to shigellosis, a serious medical problem especially among infants in developing countries. The lipopolysaccharide of S. dysenteriae is essential for virulence, and there is indirect evidence that antibodies against this O-specific polysaccharide (O-SP) are protective to the host (Taylor et al., 1993Go; Passwell et al., 2001Go). Thus, there is considerable interest in the development of an O-SP-based vaccine protective against S. dysenteriae type 1 (Robbins et al., 1992Go).

The O-SP of S. dysenteriae type 1 consists of a 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, cf. Table I) (Dmitiev et al., 1976Go). Using synthetic saccharides the antigenic determinant of the O-SP has been identified as the {alpha}-L-Rhap-(1->2)-{alpha}-D-Galp disaccharide in the case of two monoclonal antibodies, IgM 3707 E9 (Pavliak et al., 1993Go) and an IgG (Miller et al., 1996Go). More recently, the significance of each of the hydroxyl groups of the B-C epitope for the interaction with IgM 3707 E9 was elucidated, emphasizing the crucial role of the galactose residue (Mulard and Glaudemans, 1998Go; Miller et al., 1998Go). In the case of two other monoclonal IgM antibodies, a tetrasaccharide (A-B-C-D) has been found to be required for recognition (Miller et al., 1996Go). In a recent study, it has been shown that an octasaccharide [B-C-D-A']2 and longer fragments of the O-SP are efficient haptens in mice, whereas a corresponding tetrasaccharide B-C-D-A' conjugate was not immunogenic (Pozsgay et al., 1999Go). So far, however, factors such as conformational properties, which potentially determine the immunogenicity of these O-SP fragments are not properly understood. Early nuclear magnetic resonance (NMR) studies suggest that the pentasaccharide A-B-C-D-A' is the shortest saccharide fragment that shows the conformational features of the natural O-SP (Pozsgay et al., 1993Go). Moreover, NMR studies using 13C-labeled saccharides (Pozsgay et al., 1998Go) have given some additional evidence for conformational differences depending on the length of the saccharide sequence. Recently, a model of the octasaccharide [B-C-D-A']2 based on NMR data and simulated annealing has been presented (Coxon et al., 2000Go). In the present study, we report on a detailed theoretical conformational analysis using modified hard-sphere exo-anomeric (HSEA) calculations, MM3 molecular mechanics, and molecular dynamics on a series of fragments of the O-SP of S. dysenteriae type 1.



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Table I. Structure and notation of the studied saccharides

 

    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Modified HSEA calculations
Systematic conformational search using the GLYCAN program on the compounds 1–8 predicted only one favored {phi}/{psi} conformation for each of the glycosidic linkages of {alpha}-L-Rha-(1->3)-{alpha}-L-Rha (A-B), {alpha}-L-Rha-(1->2)-{alpha}-D-Gal (B-C), and {alpha}-D-GlcNAc-(1->3)-{alpha}-L-Rha (D-A'). These conformations are not significantly affected on elongation of the saccharide chain within this series of compounds (Figure 1). However, for the {alpha}-D-Gal-(1->3)-{alpha}-D-GlcNAc (C-D) linkage the favored conformation is at {phi}/{psi} {approx} –65°/–40° (I) in the di- and trisaccharides 3 and 5, whereas in the tetra- and pentasaccharides 6, 7, and 8 a second favored conformation II at {phi}/{psi} {approx} 15°/30° emerges with 8%, 9%, and 59% probability, respectively (Figure 1). Thus, these calculations indicate that the pentasaccharide 8 exists as two major conformers, I and II, distinguished by the conformation of the C-D linkage. Complete energy minimization of pentasaccharide 8 with GESA (Table II) starting from the geometries obtained with GLYCAN gives an energy difference of 1.3 kcal/mol in favor of conformation II. Conformer II shows a "hairpin" shape that is stabilized due to favorable van der Waals contacts between the rhamnose residues (A,B) at the nonreducing end and the N-acetylglucosamine (D) and rhamnose (A') at the reducing end (cf. MM3 calculations). These findings prompted a more detailed conformational analysis with MM3.



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Fig. 1. {phi}/{psi} probability diagrams for the glycosidic linkages of the trisaccharide 5 (top row), the tetrasaccharide 7 (middle row) and the pentasaccharide 8 (bottom row) based on systematic conformational search with the GLYCAN program. For each diagram the {phi}/{psi} distribution refers to the glycosidic linkage indicated by underscored residue letters, see Table I. Note that the calculations indicate a single favored conformation, I, for the {alpha}-D-Gal-(1->3)-{alpha}-D-GlcNAc (C-D) linkage in the trisaccharide, whereas in the tetra- and pentasaccharides a second peak, conformation II, emerges. All the other glycosidic linkages showed only one favored conformation within this series of fragments. White areas in the basis of the diagram indicate ranges of {phi}/{psi} torsion angles that have not been explicitly calculated due to the filtering applied (see text); light gray indicates grid cells that were calculated but found to have a probablility of < 0.1%. The dark gray bars indicate grid cells with a probability of > 0.1% according to the Boltzmann distribution at 310K.

 


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Table II. Minimum energy conformations calculated with GLYCAN/GESA and MM3

aFor complete formulae see Table I.

bResults obtained with GLYCAN/GESA are shown in italics.

cAll the conformations shown are gt with respect to the 6-hydroxyl group of the Gal and GlcNAc residues. The corresponding GlcNAc-gg conformation had slightly higher energy.

dRelative energy (kcal/mol) in relation to the lowest energy conformation found with the respective method (GESA and MM3 with {varepsilon} = 80).

eRoman numerals I and II in parentheses designate the type of conformation at the {alpha}-D-Gal-(1–3)-{alpha}-D-GalNAc linkage (see text).

 
MM3 calculations
Adiabatic potential energy maps calculated with MM3 for the constituent disaccharide units of the O-SP are shown in Figure 2. These results show only one major preferred conformational range of {phi}/{psi} torsions in the case of the disaccharides 1, 2, and 4. The low energy ranges of the two former disaccharides show a fairly wide extension in the {psi}-dimension with two minima (Table II and Figure 2), whereas disaccharide 4 is quite restricted to a single confined {phi}/{psi} energy minimum. The C-D disaccharide (3), on the other hand, shows two major minimum energy regions separated by a significant barrier. One minimum is centered at {phi}/{psi} {approx} –30°/–30° (I) and the other at {phi}/{psi} {approx} –15°/45° (II). Calculations of probabilities based on the relaxed potential energy maps for the C-D disaccharide indicate a population of 72% for conformer I and 28% for conformer II (Table III). The barrier between these energy minima is about 2.5 kcal/mol and is due to steric interference of the N-acetyl group of residue D with the 6-hydroxymethylene group of residue C. This was demonstrated by comparative calculations on the corresponding {alpha}-D-Galp–(1->3)-{alpha}-D-Glcp disaccharide, which lacks this particular energy barrier in its {phi}/{psi} potential energy map.



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Fig. 2. Adiabatic MM3 energy maps for different disaccharide moieties contained within the O-SP of S. dysenteriae type 1: A-B) {alpha}-L-Rhap-(1->3)-{alpha}-L-Rhap (1); B-C) {alpha}-L-Rha-(1->2)-{alpha}-D-Gal (2); C-D) {alpha}-D-Gal-(1->3)-{alpha}-D-GlcNAc (3); D-A') {alpha}-D-GlcNAcp-(1->3)-{alpha}-L-Rhap (4). Contour levels are shown at every 1 kcal/mol from 1 to 10 kcal with colors from blue to red. The two favored conformations I and II calculated for the C-D linkage are indicated.

 

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Table III. Probability (%) of conformations at the {alpha}-D-Gal-(1->3)-{alpha}-D-GlcNAc linkage in different saccharides, calculated from systematic search with MM3 and GLYCAN (in brackets)
 
The adiabatic {phi}/{psi} maps for the B-C and C-D linkages in the case of trisaccharide 5 are shown in Figure 3. The favored {phi}/{psi} region II of the C-D linkage is more pronounced and somewhat more extended toward positive {phi} values than for disaccharide 3. Calculation of the {phi}/{psi} probability distribution for this linkage in trisaccharide 5 (Figure 4) showed a population of 38% for conformer II. Also the passage between the two minima in the adiabatic energy map becomes more narrow than in the case of the disaccharide 3. These differences must be ascribed to the presence of the rhamnose residue B linked at position 2 of the galactose. The adiabatic map for the B-C linkage in trisaccharide 5 (Figure 3) shows a single low energy range which is restricted to {phi}/{psi} values which correspond to the secondary energy minimum of disaccharide 2 (Table II and Figure 2). These restrictions are obviously due to interference of the D residue with the B-C moiety.



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Fig. 3. Adiabatic MM3 energy maps (see text) for the B-C (left) and C-D (right) linkages in the trisaccharide 5 (top), the tetrasaccharide 6 (middle) and the pentasaccharide 8 (bottom). Note the two major minima the C-D linkage (cf. Figure 2) and the shift of minimum II toward {phi}/{psi} {approx}15°/30° in the case of the pentasaccharide. Contour levels 1–10 kcal as above. An additional contour delimits the {phi}/{psi} region, which was excluded from explicit calculation due to the filtering applied (see text).

 


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Fig. 4. The calculated {phi}/{psi} probability distributions shown as bar diagrams for the B-C (left) and C-D (right) linkages in the disaccharides 2 and 3 (top), the trisaccharide 5, the tetrasaccharide 7 and the pentasaccharide 8 (bottom). These Boltzmann distributions are based on all the minimized states obtained in the MM3 driver calculations. Conformation I and II have been indicated in the case of the C-D linkage. For an explanation of the greytones see legend of Figure 1.

 
MM3 relaxed map calculations with respect to the {phi}/{psi} torsions of the B-C-D moiety in the tetra- and pentasaccharides 6, 7, and 8 resulted in energy and probability maps for the C-D linkage (Figure 4) with increasing preference for conformation II. In particular the C-D energy map for pentasaccharide 8 showed a narrowing and shift of energy minimum II from the negative {phi} range, as seen in trisaccharide 5, to {phi}/{psi} {approx} 15°/30°, which is in agreement with the results of the systematic search with GLYCAN and GESA (Figure 1 and Table II). Calculations of {phi}/{psi} probability distributions from the MM3 results indicate a population of 83% for C-D conformation II of the pentasaccharide 8 (Figure 4 and Table III). From energy and probability {phi}/{psi} maps it is apparent that the favored {phi}/{psi} ranges of the C-D and the B-C linkages in pentasaccharide 8 are more narrow than in trisaccharide 5 and tetrasaccharide 7 (Figures 3 and 4).

Based on geometries obtained from the MM3 systematic search, complete MM3 energy minimizations with full matrix optimization were performed on the saccharides 1–8. This showed only minor geometry changes in the glycosidic {phi}/{psi} torsion angles compared with the starting geometries. The MM3 results indicate that the hairpin conformation (II) of the pentasaccharide 8 (Figure 5), with close contacts between the A-B and D-A' moieties, is 0.7 kcal/mol lower in energy than the extended conformation (I). Preliminary investigations at a dielectric constant of {varepsilon} = 4.0, emphasizing polar interactions, indicate that the hairpin conformation of the pentasaccharide is favored by 1.5 kcal/mol compared to the extended conformation. It was observed that the hairpin conformation for pentasaccharide 8 calculated with MM3 differed somewhat from the results of GLYCAN/GESA with respect to the {psi} torsion of the D-A' linkage, whereas the conformations of the A-B-C-D moiety from GLYCAN/GESA and MM3 are almost identical. This deviation of the GLYCAN/GESA results is most likely due to the rigid residue assumptions used in the modified HSEA procedure.



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Fig. 5. Relaxed stereopairs of the two major minimum energy conformations of the pentasaccharide 8 according to calculations with MM3: the extended conformer (top) with the C-D linkage in conformation I ({phi}/{psi} {approx} –40°/–30°) and the preferred hairpin conformer (bottom) characterized by C-D conformation II ({phi}/{psi} {approx} 10°/30°). Note that the orientation of the primary hydroxyl group of the galactose residue in relation to the N-acetyl group differs between these conformations. An intermediate location of the N-acetyl group would lead to collision with the primary hydroxyl group. This illustrates the "bisecting" role of the N-acetyl group on the conformational space of this {alpha}-(1->3) linkage (cf. Figures 3 and 4). Note the close contacts between the upstream A-B moiety and the downstream N-acetylglucosamine in the hairpin conformation. The geometries for the A-B-C moieties are almost identical in the two conformers. The MM3 geometries shown differ very little from the corresponding conformers obtained with GLYCAN/GESA.

 
Molecular dynamics
Molecular dynamics (MD) calculations on disaccharide 3 covering a simulation time of 2.5 ns shows two favored conformations for the C-D linkage that are in good agreement with the results of the MM3 calculations (Figure 2). In the MD simulations, conformations I and II were found to interconvert at about every 500 ps with some preference for conformer I. However, due to the low number of transitions occuring during the simulation, the relative populations of conformers I and II could not be estimated. MD simulation on trisaccharide 5, starting from the two major minimum energy conformations obtained with MM3, did not show interconversion between the conformers within the conditions of the simulation (2.5 ns, 300K). Similarly, preliminary MD simulations performed on pentasaccharide 8 indicated that the hairpin type of conformer is stable for simulation periods of 2.5 ns.

Modeling of larger fragments
Modeling of the octasaccharide 9 and the hexadecasaccharide 10 was performed on the basis of the hairpin conformation obtained with MM3 for the pentasaccharide 8. The results indicate that extensions with additional repeating units are not interfering with the preferred internal conformations of single repeating units. The modeling shows that consecutive repeating units are arranged in a left-handed helical structure with the galactose residues pointing outwards (Figure 6). Consecutive repeating units with hairpin conformation are located on opposite sides of the helix axis with an angular displacement of –169° and a rise of 8.5 Å. A single turn of the helix contains 2.12 repeating units (8.5 residues) and has a pitch of 18 Å. Comparative modeling of the oligosaccharide chain with the C-D conformation I in all the repeating units shows a flat, ribbonlike structure with about equal exposure of the galactose and the rhamnose residues. Energy data from single point minimizations with MM3 indicate that the "all hairpin" conformation of the hexadecasaccharide 10 is preferred by 3.4 kcal/mol compared to the corresponding "all extended" conformation with type I conformation. Preliminary calculations were performed at a dielectric constant of 4.0 to study the possible influence of intramolecular hydrogen bonding. These calculations give an energy difference of 7.5 kcal/mol between the all hairpin and the all extended conformations.



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Fig. 6. Top: Side view of a model of eight repeating units [A-B-C-D]8 built on the basis of minimum energy conformation II of the pentasaccharide 8 as obtained with MM3. Repeating units with haripin conformation form a left-handed helix in which the galactose residues (green) are preferentially exposed. Also rhamnose (B) and GlcNAc (D), shown in orange and gray, are partially accessible. The upstream rhamnose residue (A, violet) of each repeating unit is located within the central "core" of the helix. The galactose of the upstream repeating unit on the right side has been designated number 1 to indicate the relations to the axial view (below). Lower right: Axial view of eight repeating units in hairpin conformation obtained after rotation of the projection above by –90° about a vertical axis. The galactose residues (green, numbered from 1 to 8) protrude at the surface of the helix with an angular displacement of –169° between consecutive galactose residues. The rhamnoses A, shown in violet, are buried in the center of the helix, with limited ability to interact with a binding protein. These residues may thus be considered to have a role as "scaffold" in the helical structure.

 
Docking with antibody
The pentasaccharide 8 in its hairpin conformation was docked with the homology modeled complementarity determining region of the IgM antibody 3707 E9 (Figure 7). The orientation obtained of the B-C moiety did not differ significantly from the geometry reported earlier for the modeled complex of this antibody with the {alpha}-L-Rhap-(1->2)-{alpha}-D-Galp-OMe disaccharide (Miller et al., 1998Go). The galactose is deeply buried in the site making hydrophobic contacts with Trp L:91 and hydrogen bonds to the protein through its O6 and O3 hydroxyl groups. The adjacent rhamnose residue (B) contributes to the binding by hydrophobic contacts with Trp H:98. This is in full agreement with known binding data of natural fragments (Pavliak et al., 1993Go) and deoxy analogues of disaccharide 2 (Mulard and Glaudemans, 1998Go). The adjacent upstream rhamnose residue (A) and the N-acetyl glucosamine make weak van der Waals contacts with the protein, but no hydrogen bonds were identified. The rhamnose A' at the reducing end points out from the site and does not make any van der Waals contacts with the protein.



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Fig. 7. Model of the hexadecasaccharide 10 in hairpin conformation (II) docked with the binding site of the monoclonal antibody IgM 3707 E9 (Miller et al., 1998Go). The pentasaccharide moiety (8), shown with colored atoms, was used for the docking with the ICM program, and the hexadecasaccharide was subsequently fitted in replacing the pentasaccharide. The Rha-Gal (B-C) epitope, shown with orange and green dot surfaces, is involved in close contacts with the protein. The galactose, forming the vertex of the repeating unit, is deeply buried, making hydrophobic contacts with tryptophan L:91 and accepts a hydrogen bond at O3 while the 6-hydroxyl group acts as a hydrogen bond donor. The adjacent rhamnose (B) residue makes favorable hydrophobic contacts with tryptophan H:98. These results are in full agreement with deoxy sugar binding data (Mulard and Glaudemans, 1998Go) and the previous docking of the B-C disaccharide (Miller et al., 1998Go). The rhamnose residue (A) and the upstream repeating unit are involved in weak van der Waals contacts with the protein. The downstream GlcNAc (D) makes weak van der Waals contacts with the protein but the rhamnose (A'), and the downstream repeating units are devoid of contacts with the protein. Comparative modeling of conformation I of the oligosaccharide shows substantial steric interference between the downstream GlcNAc residue (D) and the protein.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Conformations of the {alpha}-D-Gal-(1->3)-{alpha}-D-GlcNAc linkage: the hairpin conformation
The present study indicates that two major favored conformations for the C-D linkage have to be considered for fragments of the S. dysenteriae type 1 O-antigen. For short fragments (compounds 3, 5) GLYCAN as well as MM3(96) show preference for conformation I ({phi}/{psi}{approx}–45°/–30°), albeit MM3 also shows a significant population of conformation II ({phi}/{psi}{approx}–15°/30°) (Figures 14 and Table III). The fact that two distinct conformations are calculated for the C-D linkage is basically due to the collision of the N-acetyl group of the N-acetylglucosamine residue (D) with the hydroxylmethylene group of the galactose residue (C). In the tri- and tetrasaccharides 5, 6, and 7 the flanking rhamnose residues accentuate the energy barrier between these conformations and increasingly favor conformation II. In the case of pentasaccharide 8, both GLYCAN/GESA and MM3 calculations predict that the C-D conformation II, giving rise to a hairpin conformation, is predominating (Figures 1 and 4, Table III) as a result of favorable van der Waals contacts between the upstream A-B and the downstream D-A' moieties (Figure 5). The {phi}/{psi} potential energy map for the C-D linkage of pentasaccharide 8 shows that the minimum energy well II is somewhat shifted to more positive {phi} values ({phi}/{psi} {approx} 15°/30°) corresponding to a steric strain of 1.5–2 kcal/mol for the C-D moiety according to the potential energy map of the disaccharide 3 (Figure 2). This strain is apparently outbalanced by the favorable contacts between residues at both ends of the pentasaccharide. Similarly, calculations on the longer fragments (compounds 9–10) using GLYCAN, GESA, and MM3 show a preference for conformation II of the C-D linkage in the O-SP, leading to a hairpin conformation for each repeating unit.

Our present results regarding the conformations of the C-D moiety and their influence on the overall saccharide conformation are partly supported by available NMR data. Early NMR studies indicated that fragments of the O-SP of S. dysenteriae type 1 have to be of a certain length to produce NMR signals characteristic for the native O-SP (Pozsgay et al., 1993Go; Pozsgay and Coxon, 1994Go). In particular the C-D moiety showed differences in 13C shifts and 1JC1,H-1 coupling between short fragments (compounds 3, 5, 6) and longer sequences, such as pentasaccharide 8, which were attributed to conformational differences. Later, NMR studies including analysis of 3JCH coupling (Pozsgay et al., 1998Go), indicated that on extension of the disaccharide 3 to the pentasaccharide 8 the {psi} value of the C-D linkage is changed from ± 21° to ± 31° (the signs were not determined). With respect to the disaccharide 3 the present results suggest that the experimental {psi} value of ± 21° ought to be interpreted as mainly –21° (conformation I). The experimental value of ± 31° obtained for pentasaccharide 8 is in good agreement with Boltzmann weighted averages |{psi}| {approx} 30° and 34° calculated on the basis of the present systematic search with GLYCAN and MM3, respectively.

Recently Coxon et al. (2000)Go reported on the conformation of the octasaccharide 9 as based on NOE and coupling data. The results were interpreted as two different conformations of the C-D linkage coexisting within the same octasaccharide: –72°/25° at the reducing end and 61°/30° at the nonreducing end. These {psi} values of 25° and 30° are in agreement with conformation II in the present study. However, the {phi}-values cannot be reconciled with the present calculations, especially not {phi}/{psi} = –72°/25° which is in a high energy region (Figures 2 and 3), at 15 kcal/mol above the global minimum according to single point minimizations on the trisaccharide 5. The value {phi}/{psi} = 61°/30° shows a better agreement with conformation II of the pentasaccharide 8 ({phi}/{psi} {approx} 15°/30°) but is displaced toward higher {phi} values corresponding to an MM3 energy penalty of about 3 kcal/mol above the lowest minimum. The main reason for the discrepancies appears to be the fact that Coxon and colleagues have obtained the cis configuration for the N-acetyl amide bond of the two GlcNAc residues (see Figure 5 in Coxon et al., 2000Go). In carbohydrates the N-acetyl cis configuration is usually considered to have a very minor population (1:40) compared to the trans configuration (Poppe et al., 1990Go). Possibly the cis configuration is a feature remaining from the high temperature dynamics and the annealing, during which apparently no special measures were taken to prevent trapping of the pendant groups in disfavored local energy minima. Modeling of the C-D linkage with {phi}/{psi} = –72°/25° and the N-acetyl amide in the normal trans configuration shows severe steric clashing of the acetyl with the primary hydroxyl group of the neighboring galactose residue, consistent with our MM3 calculations. However, modeling of the C-D linkage with the same {phi}/{psi} conformation and the N-acetyl amide in the cis configuration indicates that this steric clash is avoided, which can explain that it was accepted as a favored conformation in the NMR-based simulated annealing. Furthermore it is notable that Coxon et al. (2000)Go did not find evidence for an extended conformation similar to our conformation I ({phi}/{psi} {approx} –45°/–30°), which is significant in our calculations and also observed by NMR and x-ray for the closely related sequence {alpha}-D-GalNAc-(1->3)-ß-D-GalNAc of the Forssman saccharide (Grönberg et al., 1994Go; Hamelryck et al., 1999Go). Whether the lack of conformations at {phi}/{psi} {approx} –45°/–30° is due to the mentioned cis configuration of the N-acetyl groups or if it is caused by the application of a single NMR restraint at {psi} = +33°, excluding the corresponding {psi} {approx} –30° range (cf. Pozsgay et al., 1998Go), is not clear from their study (Coxon et al., 2000Go).

With respect to the D-A' linkage there is also a discrepancy between the results of the NMR- based simulated annealing and our MM3 modeling. We get a single major well-defined minimum at {phi}/{psi} {approx} –40°/-40° with MM3, but Coxon et al. (2000)Go describe two conformations at –35°/42° and –74°/–31°. This discrepancy might also be related to the cis configuration of the N-acetyl groups. However, with respect to the other {phi}/{psi} torsions of the octasaccharide, that is, the B-C and A-B linkages, there is good agreement between the NMR study (Coxon et al., 2000Go) and our results. Regarding the B-C linkage there is also good agreement with an NMR study on the disaccharide 2 (Coxon et al., 1997Go). Furthermore, the present results on the A-B linkage are supported by earlier studies on this type of linkage in a streptococcal polysaccharide (Kreis et al., 1995Go; Stuike-Prill and Pinto, 1995Go).

From our present study it is clear that filtered systematic search with modified HSEA (GLYCAN) and MM3 are powerful methods for the exploration of the conformational space of oligosaccharides (cf. Figures 14). Corresponding investigations with MD tend to be biased by the choice of the starting geometry if there are significant energy barriers between the favored conformations (Carver et al., 1990Go). Moreover, the excellent computational scalability of the systematic search on multiprocessor systems make these methods of interest as an alternative to MD for medium sized saccharides (three to five residues). Thus, the filtered systematic search, especially with a well-established force field like MM3, should provide information of value for the evaluation of NMR data.

The helical structure of the O-SP and epitope recognition
For longer sequences the calculated hairpin conformations of several repeating tetrasaccharide units result in helical structures in which the galactose residues (C) are preferentially exposed with some exposure also of the adjacent rhamnose (B) and N-acetyl glucosamine (D) residues (Figures 5 and 6). This structure is in agreement with binding studies that have shown that the galactose together with the neighboring rhamnose constitute the major antigenic determinant of this O-SP in the case of the monoclonal antibodies IgM 3707 E9 (Pavliak et al., 1993Go) and IgG 5338 H4 (Miller et al., 1996Go). The successful docking of the hairpin conformer of pentasaccharide 8 with the IgM antibody binding site (cf. Figure 7) was in full agreement with binding studies on modifications of disaccharide 2 (Mulard and Glaudemans, 1998Go) and the previous docking of disaccharide 2 (Miller et al., 1998Go). This supports our interpretation that the hairpin conformer is essential for antibody recognition in the case of IgM 3707 E9. It is notable that the B-C-D moiety, contained in repeating units with hairpin conformation, is conformationally very well defined due to the restricted flexibility of the B-C and C-D linkages, as shown for the pentasaccharide 8 (Figures 3 and 4). Probably this restricted flexibility of the exposed epitope gives rise to a favorable entropic contribution to the free energy of binding (cf. Navarre et al., 1999Go).

The upstream rhamnose residue (A) in each repeating unit is located centrally in the hairpin helix (Figure 6) and is thus largely shielded from protein interactions. This implies that this rhamnose is not essential for binding per se but rather has a role as a "scaffold" in the O-SP. Although this appears plausible in the case of IgM 3707 E9 and IgG 5338 H4, it is somewhat contradicted by the fact that some IgG antibodies require the entire tetrasaccharide moiety A-B-C-D for binding (Miller et al., 1996Go). The explanation could be that the A residue contributes by stabilizing conformation II of the repeating unit.

Comparative modeling of the oligosaccharide chain with conformation I of the C-D linkages shows a flat ribbonlike structure with about equal exposure of the galactose (C) and the adjacent rhamnose residue. Modeling of the repeating unit with this conformation in the binding site of the antibody showed substantial steric interference of the N-acetyl group of the D residue with the protein. Therefore, we conclude that the antibody IgM E3707 E9 only recognizes the hairpin conformation of the repeating unit.

The present results indicate that the conformational preferences of the B-C-D moiety are essentially unchanged when the saccharide is extended beyond the length of the pentasaccharide 8. This is in agreeement with NMR data showing that pentasaccharide 8 is sufficient to produce NMR shifts for the B-C-D moiety typical for those of the native O-SP (Pozsgay et al., 1993Go). Although the IgM 3707 E9 recognizes the disaccharide 2, we cannot exclude the possibility that the presently studied system exhibits a "conformational epitope" (Zou et al., 1999Go) in reactions with certain other antibodies (cf. Miller et al., 1996Go). Most probably such a conformational epitope is fully attained with the pentasaccharide 8. In this respect the presently studied O-SP differs from the type III group B Streptococcus capsular polysaccharide, in which several repeating units are required to give rise to the conformational epitope (Zou et al., 1999Go). With respect to immunogenicity, it has been shown that there is a significant length dependence in the case of fragments of the S. dysenteriae type 1 O-SP (Pozsgay et al., 1999Go). A minimum of two repeated B-C-D-A' units was required to elicit IgG formation in mice. Whether this is due to a conformational effect or other factors, for instance, differences in antigen processing or requirements for multivalency, is not known.

The present results suggest that a hairpin conformation of the repeating unit is involved in antibody recognition and possibly also in eliciting antibody responses. To address these questions it is of interest to design short fragments of the O-SP that are conformationally locked in a hairpin conformation by covalent links and use these for future antibody binding and immunogenicity studies.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The saccharides (1–10) studied in the calculations are listed in Table I. The notation used for the torsion angles in the glycosidic linkages is {phi} = H1 – C1 – O1 – CX and {psi} = C1 – O1 – CX – HX with X = 2 and 3, respectively, in the studied 1->2 and 1->3 glycosidic linkages. Initial calculations, carried out with the GESA (Paulsen et al., 1984Go) and GEGOP programs (Stuike-Prill and Meyer, 1990Go), indicated that the repeating unit could adopt a sharply bent structure, which in larger fragments gives rise to close contacts between remote residues. This suggested a need for a thorough conformational search, which was performed using GLYCAN as well as MM3.

The GLYCAN program, which is an in-house development based on the GESA program, is well suited for rapid screening of the conformational space of medium-sized oligosaccharides using the standard HSEA energy functions (Lemieux et al., 1980Go) with revised parameters for the exo-anomeric effect (Wiberg and Murcko, 1989Go) as implemented in GEGOP. GLYCAN initially calculates the {phi}/{psi} potential energy maps of all the types of disaccharide moieties occuring in the oligosaccharide. The complete permutation of torsion angles for the oligosaccharide is then performed only on {phi}/{psi} conformations with energies below a certain cutoff, typically 12 kcal/mol above the global minimum for each disaccharide. The hydroxymethylene group was approximated as a methyl group. For a pentasaccharide with eight degrees of freedom considered at a step length of 15°, this filtering reduced the number of conformations to be evaluated to about 0.02% of all the theoretically possible conformations. The remaining conformations (18 x 106) could be calculated in about 7 h on a 800 MHz PC using a highly optimized energy routine. Selected low-energy conformations from the GLYCAN calculations were subjected to complete energy minimizations with the GESA program with revised exo-anomeric parameters (Wiberg and Murcko, 1989Go).

MM3 (Allinger et al., 1989Go, 1990; Lii and Allinger, 1991Go) is considered to be one of the very best force fields for use with oligosaccharides (Perez et al., 1998Go; Haxaire et al., 2000Go) but has the disadvantage that calculations on larger systems are fairly time-consuming. The MM3(96) was used to calculate relaxed potential energy {phi}/{psi} maps using driver option 4 for each of the four different disaccharide units occuring in the O-SP. Different combinations of starting conformations for the primary and secondary hydroxyl groups were considered as described by French et al. (1990)Go and the lowest energy value obtained at each {phi}/{psi} point was used in the generation of adiabatic potential energy {phi}/{psi} maps with a step length of 15°. In the case of trisaccharide fragments 4D MM3 driver calculations were carried out for the {phi}/{psi} torsions of the constituent glycosidic linkages. The input for these calculations was generated with an in-house developed program selecting {phi}/{psi} points within a range of 12 kcal/mol from the global minimum of the MM3 energy map of the corresponding disaccharide unit. In the case of the trisaccharide 5 permutation resulted in 69,360 starting conformations. Adiabatic energy maps were generated considering also the four different combinations of favored rotamers for the primary hydroxyl groups. Due to limitations in computer resources it was, however, not possible to perform a full {phi}/{psi} systematic search with MM3 for the tetra- and the pentasaccharides (6, 7, and 8). The B-C-D moiety, which is most critical for the overall shape of these saccharides, was analyzed at the usual step length of 15° and the third linkage was stepped with lower resolution to improve the conformational search with respect to the B-C-D moiety. Only conformations of the trisaccharide 5 with energies within 12 kcal/mol from the lowest minimum were considered as starting geometries (24,187 conformations). In the case of the tetrasaccharides the linkages to the fourth residue, D-A' and A-B of compounds 6 and 7, respectively, were sampled at seven points, including the major minima and evenly dispersed points on the 1 kcal/mol contour curve of the corresponding {phi}/{psi} map. Due to limitations in processor time, only the gt conformation of the primary hydroxyl groups of residues B and C could be considered. However, runs on the trisaccharide 5 had shown that this arrangement of the primary hydroxyl group is significantly favored. The pentasaccharide 8 was studied with MM3 using the same 6D stepping as in the case of tetrasaccharide 7, but leaving the D-A' linkage at the reducing end for free minimization starting from its favored conformation obtained from runs on shorter fragments. Local minima found in the driver calculations were subjected to complete minimizations using MM3 full-matrix minimization. The resulting true local minimum points were then fed into Sybyl program (Tripos, St. Louis, MO) and subjected to cluster analysis using the Selector module. The present MM3 calculations were performed with a dielectric constant {varepsilon} of 80 to simulate an aqueous medium (cf. Haxaire et al., 2000Go). In a few explicitly stated cases {varepsilon} = 4 was used to evaluate the possible effects of intramolecular polar interactions.

The calculations were carried out on three to four computers with dual Intel 500 MHz Celeron processors using the Linux version of MM3(96) program (QCPE, Bloomington, IN). The CPU time required for the MM3 calculations on pentasaccharide 8 was about 1300 h. Both the output from the GLYCAN systematic search and the MM3 relaxed potential energy maps were used to calculate Boltzmann probability distributions at a temperature of 310K. The energy contour maps and the probability diagrams were plotted using the GSHARP program (AVS Inc, Waltham). Molecular dynamics calculations were carried out with AMBER 5 (Case et al., 1997Go) using the glycam parameters (Woods et al., 1995Go) according to the release glycam41_98a.

The pentasaccharide 8 was docked with the binding site of the IgM 3707 E9 antibody modeled from crystal structures of homologous immunoglobulins as described earlier (Miller et al., 1998Go). The light chain of 3707 E9 showed a strong homology with the IgG HC19 (PDB code 1GIG), and the first and second hypervariable loops of the heavy chain showed homology with IgA J539 (PDB code 2FBJ). Therefore no loop modeling was performed for these regions. The third hypervariable loop of the heavy chain of 3707 E9 was modeled from the heavy chain of the IgG 26–10 Fab-digoxin complex (PDB code 1IGI) with manual adjustment of two sequential prolines and subsequent energy minimization and conformational search using the ICM program (Abagyan et al., 1994Go). The docking was performed using a Monte Carlo algorithm of the ICM program with random positioning of the ligand followed by minimization in which both the ligand and the side chains were allowed to be flexible. Weak tethering was applied between the galactose and the tryptophanes (L:91 and H:33) in the proposed site. The partial charges of the pentasaccharide ligand were determined by the method of Gasteiger and Marsili, 1980Go.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
B. Meyer, University of Hamburg, is gratefully acknowledged for versions of the GESA and GEGOP programs. The authors wish to thank I. Pascher for critical comments and S. Sundell for maintaining the Linux computers. The GLYCAN program can be made available from the author (P.G.N.). This work was supported by a grant from the Mizutani foundation for Glycosciences and from the Swedish Medical research council (grant no. 006).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
HSEA, hard-sphere exo-anomeric; MD, molecular dynamics; NMR, nuclear magnetic resonance; O-SP, O-specific polysaccharide.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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