Specific interactions between carbohydrate-binding modules and cellulose

Polysaccharidases and the carbohydrate-binding modules (CBMs) have evolved to mirror the diversity inherent in the crystalline and amorphous structures of cellulose and other polysaccharides abundant in nature. In this study, the binding specificities for cellulose of CBMs representing each of the three functional types of CBMs (types A, B and C), were determined by a series of pair-wise competition binding experiments. These studies show that different binding sites of phosphoric acid-swollen cellulose (PASC) are bound by different CBMs: type A localize to crystalline regions, type B localize to the amorphous regions. The data suggest that there is heterogeneity in the amorphous regions of PASC. The binding of the family 2a CBM from Cellulomonas fimi xylanase 10A (CBM2a) to insoluble cellulose was characterized in more detail. Competition experiments indicate that CBM2a binds to the crystalline regions of cellulose. It has been reported that CBM2a binds irreversibly to cellulose however, it is shown here that CBM2a bound to the cellulose surface can be displaced by CBM2a in solution. CBM2a has a number of conserved residues on the binding face, including three tryptophans that have been implicated in the binding reaction. Site-directed mutation and a Langmuir-type adsorption isotherm analysis was used to determine the individual contributions of W17, W54, W72 and a number of neighbouring residues to the overall binding affinity of CBM2a for cellulose. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. In an attempt to formulate a structural model of CBM2a bound to the surface of cellulose, observation of ¹⁵N-labelled CBM2a bound to ¹³C-enriched cellulose using solid state NMR was attempted. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, a model is proposed for the interaction between the polypeptide and the crystalline surface that is also likely to apply to other type A CBMs.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat