Conformational Studies of the O-specific Polysaccharide of Shigella flexneri 5a and of Four Related Synthetic Pentasaccharide Fragments Using NMR and Molecular Modeling

Abstract

As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic pentasaccharide fragments. The NMR study, based on the analysis of 1 H and 13 C chemical shifts, the evaluation of inter-residue distances, and the measurement of oneand three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution. Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S. flexneri 5a has a greater affinity for this pentasaccharide than for the others. We carried out a complete conformational search on the pentasaccharides using the CICADA algorithm interfaced with MM3 force field. We calculated Boltzmann-averaged inter-residue distances and 3 J C,H coupling constants for the different conformational families and compared the results with NMR data for all pentasaccharides. Our experimental data are consistent with only one conformational family. We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS. The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.

Structures of synthetic O-antigen fragments from serotype 2aShigella flexneriin complex with a protective monoclonal antibody

“…S4), showing that both observed AB conformations in the two serotypes are favorable. Similarly, the ␣-D-Glcp(E)-(1-Ͼ3)-␣-L-Rhap(B) linkage of the 5a serotype is not sterically compatible with the Y serotype structure, as has been previously noted (24). The pattern of serotype glucosylation can therefore impose constraints on the structure of extended O-Ag chains.…”

“…The model polysaccharide chain thus obtained is a right-handed helix of pitch Ϸ23 Å, diameter Ϸ15 Å, and nearly three RUs per turn. Interestingly, the helix has similar parameters to the model proposed for serotype 5a O-Ag, which was based on NMR data and modeling studies (24). But unlike the serotype 5a model, where Glc(E) protrudes outwards perpendicular to the helical axis in a solvent-exposed orientation, Glc(E) in the serotype 2a is folded under the backbone at approximately the same radial distance from the helical axis as the residues ABCD.…”

Toward a Better Understanding of the Basis of the Molecular Mimicry of Polysaccharide Antigens by Peptides

“…In addition, inter-residue 1 H-1 H distances were also calculated from a trROESY spectrum obtained with a mixing time of 400 ms to take spin diffusion into account, if any. Comparison of these distances with those measured for unbound DA(E)BC-OMe (33) suggested that the pentasaccharide conformation was not significantly modified upon binding to the mIgAs (Table 5).…”

“…Clustering of solutions was done by root mean square fitting (Ͻ1 Å). The best solution of each cluster was used to propagate the helices to 20 residues while keeping the conformations determined previously (33). Twenty different conformers of the p100c peptide were also docked in the mIgA I3 Fab-binding site using the rigid body approach of the Autodock3 program.…”

“…Study of both the antigenicity and the conformation of the four synthetic frame-shifted pentasaccharides corresponding to pentasaccharide I (32) suggested that the DA(E)BC sequence is the structure that best mimics the native O-SP antigen (33). More recently, the pentasaccharide DA(E)BC was shown to act as a mimotope.…”