Site-directed mutagenesis and sugar-binding properties of the wheat germ agglutinin mutants Tyr73Phe and Phe116Tyr.
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 Phe116-->Tyr (domain C), were produced. The binding activity for (GlcNAc)3 and the three-dimensional structure of these mutants were characterized by comparing with the properties of wild-type WGA2. Equilibrium dialysis experiments using (GlcNAc)3 indicated that the mutation Tyr73-->Phe reduced the overall sugar-binding activity at both pH 5.9 and pH 4.7. In addition, positive cooperativity toward (GlcNAc)3 binding was observed at pH 4.7. In contrast, the mutation of Phe116-->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 Glu115 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 Phe116-->Tyr caused the formation of a new hydrogen bond between Tyr116 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.
Nagahora
H
H
Institute of Biological Science, University of Tsukuba, Japan.
Harata
K
K
Muraki
M
M
Jigami
Y
Y
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D050260:Carbohydrate Metabolism; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D014909:Wheat Germ Agglutinins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D014909:Wheat Germ Agglutinins