Molecular Recognition of Oligosaccharide Epitopes by a Monoclonal Fab Specific for Shigella flexneri Y Lipopolysaccharide:  X-ray Structures and Thermodynamics^,

Abstract

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.

Structural basis of peptide–carbohydrate mimicry in an antibody-combining site

“…The crystals were frozen by being placed in nitrogen gas stream cooled at Ϫ170°C. Initial proof of the binding of the octapeptide was provided by its well resolved density in a 3-Å resolution difference Fourier (F o Ϫ F c ) map calculated with the sugar-free Fab structure phases (11). The intensity data set was collected by using DIP2030 (MacScience, Yokohama, Japan) area detector mounted on a RU200 rotating anode (Rigaku, Tokyo, Japan).…”

“…The CNS suite of programs (15) was used in the structure determination and refinement ( Table 1). The structure of the Fab-peptide complex was initially refined by the rigid-body method by using the unliganded coordinates of native Fab (11) and data to 3 Å. The density of the octapeptide in the antigenbinding site is clearly evident in the (F o Ϫ F c ) and (2F o Ϫ F c ) Fourier maps, allowing easy fitting of its known sequence.…”

“…1 Titration Microcalorimetry. Calorimetric measurements were made by using the Microcal MCS titration microcalorimeter (Microcal, Northampton, MA) and following a similar procedure and data analysis used in the binding of oligosaccharide antigen to SYA͞J6 antibody (11). The concentration of the antibody was measured by UV and that of the peptide was calculated based on quantitative amino acid analysis composition.…”

“…We chose to study the anti-Shigella monoclonal antibody SYA͞J6 (10) Fab complexed with a peptide mimetic, because the corresponding structures and thermodynamics of complexes with carbohydrate O-antigens were recently available for comparison (11). SYA͞J6 is a murine IgG 3 ( ) antibody specific for the polysaccharide O-antigen of the Shigella flexneri Y lipolysaccharide.…”

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…an extended binding site to accommodate long sugar chains) are shared with proteins of clinical relevance (e.g. antibodies [78,79] and viral proteins [80]) making the study of CBMs relevant beyond the recognition of plant cell-wall polysaccharides.…”