The Conformation of the T-antigen Disaccharide Bound toMaclura pomifera Agglutinin in Aqueous Solution

Abstract

The complex of Maclura pomifera agglutinin with the T-antigen disaccharide (␤-D-Gal-(133)-␣-D-GalNAc-(13O)-Me) was investigated by NMR spectroscopy in aqueous solution. Intramolecular transferred nuclear Overhauser enhancement (NOE) effects between the monosaccharide moieties were used to derive the ligand conformation in the lectin-bound state. Ligand protons in contact with the protein were identified by saturation transfer difference experiments and intermolecular transferred NOE effects. It is demonstrated that structural differences exist for the ligand-lectin complex in aqueous solution as compared with the previously published crystal structure (Lee, X., Thompson, A., Zhiming, Z., Ton-that, H., Biesterfeldt, J., Ogata, C., Xu, L., Johnston, R. A. Z., and Young, N. M. (1998) J. Biol. Chem. 273, 6312-6318). In order to accommodate the O-methyl group of the disaccharide, the amino acid side chain of Tyr-122 has to rotate from its position in the crystal. The NMR data are in accord with two conformational families at the ␤-(133)glycosidic linkage in the solution complex with interglycosidic angles / ‫؍‬ 45/؊65°and ؊65/ ؊18°. These differ from the bound conformation of the ligand in the crystal ( / ‫؍‬ 39/؊8°) and are not highly populated by the ligand in the free state. The reason for the structural differences at the ␤-(133)glycosidic linkage are hydrogen bonds that stabilize the relative orientation of the monosaccharide units in the crystal. Our results demonstrate that the crystallization of a protein-carbohydrate complex can interfere with the delicate process of carbohydrate recognition in solution.The core 1 disaccharide 1, ␤-D-Gal-(133)-␣-D-GalNAc, Scheme 1, is O-glycosidically linked to serine or threonine, a major component of anti-freeze glycoproteins (2) and mucins (3). Associated with carcinoma, it is one of the best characterized carbohydrate tumor markers (4) called Thomson-Friedenreich or T-antigen (5, 6). Some plant lectins with a high specificity toward the T-antigen structure have the potential to be used as specific diagnostic probes for carcinoma detection. These are peanut agglutinin (7), Amaranthus caudatus agglutinin (8), and two lectins from the plant family of Moraceae, jacalin (9) and Maclura pomifera agglutinin (MPA). 1 The crystal structure of the reducing T-antigen disaccharide 2 in complex with MPA was recently solved to a resolution of 2.2 Å (1) and displays an interesting binding mode of the disaccharide. The reducing GalNAc moiety resides in the binding site of the lectin, closely surrounded by four aromatic amino acids that form a hydrophobic pocket around the ligand. The N-acetyl group as well as the Gal unit are not in direct contact with the protein. With the exception of one Asp side chain, the H-bonds to the ligand all involve atoms of the peptide backbone or water molecules. This recognition of the reducing sugar unit seems to be a specific feature of the lectins from the Moraceae family since the closely related jacalin binds methyl-␣-D-galactopyranoside in the same ...

Crystal Structure of the Jacalin–T-antigen Complex and a Comparative Study of Lectin–T-antigen Complexes

“…The difference in glycosidic torsion angles observed in solution and in the crystals has been attributed to crystal packing in MPA. 32 However, the fact that nearly the same conformation is observed in all the lectin complexes, in spite of widely different environments, appears to suggest that it corresponds to the intrinsic conformational propensity of T-antigen.…”

“…A recently reported NMR spectroscopic study on the MPA -T-antigen complex suggests two different conformations with f/c combinations of 45/ 2 658 and 2 60/ 2 188 for the bound sugar in solution. 32 Neither of the conformations is favoured in free state or observed in crystal structures. Simple modelling shows that in the jacalin -T-antigen complex f/c values 45/ 2 658 lead to a severe steric clash of Gal with the side-chain of the binding site residue Asp125 and a conserved water (W289), which is of crucial importance for sugar binding to the lectin.…”

NMR Studies of Carbohydrates and Carbohydrate-mimetic Peptides Recognized by an Anti-Group B Streptococcus Antibody

“…The combination of STD-NMR epitope mapping data with knowledge of the bound conformations of ligands, which may be obtained by trNOESY experiments, is a powerful method to build up models of antibody-ligand interaction (37)(38)(39)(40)(41)(42)(43). Therefore, trNOESY experiments (24 -28) were used to investigate the bound conformation of the peptide.…”

NMR‐Guided Molecular Docking of a Protein–Peptide Complex Based on Ant Colony Optimization

“…In contrast, our new approach is fully automated, fast, and unbiased. To demonstrate the general applicability of the docking protocol developed for the SM3-antigen complex, it was also applied to determine the complex of Maclura pomifera agglutinin with the T-antigen dis- [33] and calcium-bound S100B protein in complex with inhibitor SBi279. [34] Weimar et al studied the first complex with trNOE and STD NMR so that the same information as for the SM3 complex is available.…”

“…[34] Weimar et al studied the first complex with trNOE and STD NMR so that the same information as for the SM3 complex is available. [33] For S100B, only STD spectra were measured. [34] Results and Discussion…”