THE CRYSTAL STRUCTURE OF THE DOLICHOS BIFLORUS SEED LECTIN IN COMPLEX WITH BLOOD GROUP A TRISACCHARIDE

Thomas W. Hamelryck¹, Julie Bouckaert¹, Remy Loris¹, Lode Wyns¹ , Marilynn E. Etzler²

¹ Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
² Section of Molecular and Cellular Biology, University of California, Davis,CA 95616, USA

Keywords : protein crystallography, legume lectins, aromatic stacking, DBL

The legume lectins are a family of sugar binding proteins found in plants belonging to the Leguminoseae family. They have been used for decades as a model system for the study of protein-sugar interactions (1). The legume lectins can be subdivided in five groups according to the specificity of their structurally conserved monosaccharide binding site : Fuc specific, GlcNAc specific, Glc/Man specific, Gal/GalNAc specific, and those with a complex specificity. Crystal structures of sugar complexes are available for legume lectins with Man/Glc, Gal/GalNAc and complex specificity. These complexes indicated that the legume lectins confer affinity via four conserved residues (Asp, Asn, Gly and an aromatic residue) while a variable loop confers specificity. Structural data is still lacking on Fuc and GlcNAc binding legume lectins, but it is expected that these lectins adopt a similar strategy for sugar binding.

Recently, we determined the crystal structure of a legume lectin from the seeds of the horse gram (Dolichos biflorus, DBL) with a rather unique specificity (2). Although many GalNAc binding legume lectins have been described that also bind Gal, DBL is the only known legume lectin that binds GalNAc but not Gal. In addition, the blood group A trisaccharide (GalNAc(a1-3)[Fuc(a1-2)Gal) inhibits agglutination by DBL slightly better than GalNAc, and the Forssman disaccharide (GalNAc(a1-3)GalNAc) sixty times better. To study the specificity of DBL in molecular detail we solved the structure of DBL in complex with the blood group A trisaccharide at a resolution of 2.7 A by X-ray crystallography.

Although the blood group A trisaccharide binds to DBL with a four times higher affinity than GalNAc, only the GalNAc residue of the trisaccharide is visible in the electron density, which indicates that the other two residues are not tightly bound to the lectin. However, the structure allowed us to determine the molecular basis of the strict GalNAc specificity of DBL: the conserved aromatic residue (one of the four conserved sugar binding residues in the legume lectins) is replaced by a Leu (Leu 127) residue, thereby abolishing Gal binding. GalNAc is still capable of binding because the N-acetyl group compensates for the loss of aromatic stacking by a hydrogen bond with Gly 103 and a hydrophobic contact with Trp 132 and Tyr 104. These conclusions are backed up with site directed mutagenesis experiments in which the Leu 127 residue was changed to a Phe residue : strikingly, the mutant protein shows affinity towards both Gal and GalNAc.

The DBL structure clearly shows the importance of aromatic stacking against Gal and provides the first structure of a legume lectin in with a GalNAc residue bound in the monosaccharide site.

1. Loris, R., Hamelryck, T., Bouckaert, J., and Wyns, L. (1998) Biochem. Biophys. Acta, 1383, 9-36
2. Etzler, M.E. and Kabat, E.A. (1970) Biochemistry, 9, 869-877