Design of a pH-dependent cellulose-binding domain

Protein-carbohydrate interactions typically rely on aromatic stacking inter actions of tyrosine, phenylalanine and tryptophan side chains with the suga r rings whereas histidine residues are rarely involved. The small cellulose -binding domain of the Cel7A cellobiohydrolase (formerly CBHI) from Trichod erma reesei binds to crystalline cellulose primarily using a planar strip o f three tyrosine side chains. Binding of the wild-type Cel7A CBD is practic ally insensitive to pH, Here we have investigated how histidine residues me diate the binding interaction and whether the protonation of a histidine si de chain makes the binding sensitive to pH, Protein engineering of the Cel7 A CBD was thus used to replace the tyrosine residues in two different posit ions with histidine residues. All of the mutants exhibited a clear pH-depen dency of the binding, in clear contrast to the wild-type. Although the bind ing of the mutants at optimal pH was less than for the wild-type, in one ca se, Y31H, this binding almost reached the wild-type level. (C) 1999 Federat ion of European Biochemical Societies.