Site‐Directed Mutagenesis and Sugar‐Binding Properties of the Wheat Germ Agglutinin Mutants Tyr73Phe and Phe116Tyr

Abstract

Wheat germ agglutinin is a dimeric lectin composed of two identical subunits. Each subunit consists of four homologous hevein-like domains of 42 or 43 amino acids each. Amino acid residues at the same position in each domain involved in sugar binding are thought to play a similar role in sugar binding. In order to clarify the role of the amino acid residue at domain position 30 of wheat germ agglutinin isolectin 2 (WGA2) in sugar binding, two WGA2 variants each containing a mutation, either Tyr73-Phe (domain B) or Phell6-Tyr (domain C), were produced. The binding activity for (GlcNAc), and the three-dimensional structure of these mutants were characterized by comparing with the properties of wild-type WGA2. Equilibrium dialysis experiments using (GlcNAc), indicated that the mutation Tyr734Phe reduced the overall sugar-binding activity at both pH 5.9 and pH 4.7. In addition, positive cooperativity toward (GlcNAc), binding was observed at pH 4.7. In contrast, the mutation of Phell6-Tyr increased the overall sugar-binding activity at pH 5.9, but reduced this activity at pH 4.7 without changing the number of sugar-binding sites. Positive cooperativity was not observed at pH 5.9 or pH 4.7. X-ray crystallographic analysis of mutant WGA2 revealed that the mutation of Tyr73-Phe caused a side chain movement of the Glul15 residue of the opposite subunit that formed a hydrogen bond with Tyr73 in wild-type WGA2. No changes were observed in the backbone structure and the disposition of the benzene ring of Phe73. The mutation Phel16-+Tyr caused the formation of a new hydrogen bond between T y r l l 6 and Glu72 of the opposite subunit. The changes in the sugar-binding properties in WGA2 mutants are discussed in relation to the structural change at the binding site.

Folding and Homodimerization of Wheat Germ Agglutinin

“…Under such conditions, a complex equilibrium is taking place between ligand binding and lectin dimerization. Other binding studies on WGA (16,(42)(43)(44)(45)(46)(47)(48) used lectin concentrations similar to or even smaller than those used by Bains et al It follows then that a reexamination of the sugar-binding properties of WGA is needed.…”

Protein-Carbohydrate Interactions

“…The similarities between the binding sites lie in three tyrosine residues and a serine from one subunit while variability between the binding sites is seen in interaction involving residues from the other subunit (Wright, 1990;Nagahora et al, 1995). A different binding site was observed in the crystal structure o f the galactose specific lectin Jacalin complexed with M eaGal (Sankaranarayanan et al, 1996).…”

Identification of a carbohydrate‐binding site in physarum haemagglutinin I

“…Mechanisms for carbohydrate recognition have been studied in several lectins (1). Recently, x-ray crystallographic studies and mutational studies have demonstrated that several amino acid residues play key roles in lectin-carbohydrate interaction (2,3). Physarum polycephalum produces two types of lectins, termed haemagglutinins I and II, which have different molecular masses and carbohydrate specificities, but have relatively high afffmity for thyroglobulin (4).…”