Mutational studies of the amino acid residues in the combining site of Erythrina corallodendron lectin.

High-resolution X-ray crystallography of the complex of the Gal/GalNAc-specific Erythrina corallodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide. The contribution of these amino acids to the binding of different monosaccharides and oligosaccharides by the lectin was examined by site-directed mutagenesis. Replacement of Phe131, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity. Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active. None of the active mutants interacted with mannose or glucose. Thus, contrary to an earlier assumption. Ala218 is not responsible for the inability of E. corallodendron lectin to bind these sugars. Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data. Instead, our data suggest that Gln219 assists in the ligation of N-acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide. Comparison with other legume lectins specific for mannose/glucose, galactose, N-acetylgalactosamine, L-fucose or N-acetylglucosamine, shows that only three of the combining site residues of E. corallodendron lectin occupy invariant positions both in their primary and tertiary structures. These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E. corallodendron lectin, and an aromatic residue, either phenylalanine (as Phe131 in this lectin), tyrosine or tryptophan. We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity.