Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues

Abstract

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. 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. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…While the prevalence of aromatic amino acid residues in the binding sites of these CBMs is consistent with the majority of carbohydrate-binding proteins, the flat or platform-like binding sites are not (Figure 1; also see Figure 4A). The planar architecture of the binding sites is thought to be complementary to the flat surfaces presented by cellulose or chitin crystals [52,53]. Indeed, there has been considerable controversy regarding the location of the Type A CBM binding site in crystalline cellulose.…”

“…Tormo et al [52] proposed that the binding site comprises the hydrophobic 110 face. McLean et al [53], however, argued that, in perfect cellulose crystals, the surface area presented by the 110 face is too small to account for the binding capacity of CBMs for this ligand, prompting the authors to propose that the binding sites are more likely to comprise the hydrophilic 110 and 010 faces. A recent seminal study by Lehtio et al [54] has used transmission electron microscopy to probe the location of the CBM-binding site on Valonia crystalline cellulose.…”

“…In Type A CBMs, mutation to alanine of polar residues predicted to make direct hydrogen bonds with the crystalline polysaccharide ligands has little effect on affinity, suggesting that, in these proteins, hydrogen bonds play only a minor role in ligand recognition [53]. In Type B and Type C CBMs, replacement of direct hydrogen-bonding resides with alanine can lead to significant losses in affinity from 2-fold [58,60,71] to complete abrogation of binding [59].…”

Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

“…Cex and the papain-cleaved product, CexCD (residues 1-315), were expressed and purified by cellulose affinity chromatography, according to previously published protocols (34,35). CexCBD (residues 336 -443) was expressed directly using the plasmid pTug-KH 6 -IEGR-CBM2a in E. coli BL21 (DE3) cells (36,37). The N-terminal His 6 tag was not removed after purification with Ni ϩ2 affinity chromatography.…”

The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules

“…Such an arrangement implies that, in perfect cellulose crystals, the surface area of the proposed binding site for the CBMs is very limited (6). For this reason, other possible binding sites for the CBMs have also been contemplated (13). However, previous observations made with electron microscopy (EM) indicate that the corners are often rounded because the chains with fewest interactions with the underlying body of the crystal are easily dissociated (14).…”