Scaffoldin-borne family 3b carbohydrate-binding module from the cellulosome of Bacteroides cellulosolvens: structural diversity and significance of calcium for carbohydrate binding

The potent cellulose-binding modules of cellulosomal scaffoldin subunits belong to the greater family of carbohydrate-binding modules (CBMs). They have generally been classified as belonging to family 3a on the basis of sequence similarity. They form nine-stranded β-sandwich structures with jelly-roll topology. The members of this family possess on their surface a planar array of aromatic amino-acid residues (known as the linear strip) that form stacking interactions with the glucose rings of cellulose chains and have a conserved Ca²⁺-binding site. Intriguingly, the CBM3 from scaffoldin A (ScaA) of Bacteroides cellulosolvens exhibits alterations in sequence that make it more similar to the CBMs of free cellulolytic enzymes, which are classified into CBM family 3b. X-ray structural analysis was undertaken in order to examine the structural consequences of the sequence changes and the consequent family affiliation. The CBM3 crystallized in space group I4₁22 with one molecule in the asymmetric unit, yielding diffraction to a resolution of 1.83 Å using X-ray synchrotron radiation. Compared with the known structures of other scaffoldin-borne CBMs, a sequence insertion and deletion appear to compensate for each other as both contained an aromatic residue that is capable of contributing to cellulose binding; hence, even though there are alterations in the composition and localization of the aromatic residues in the linear strip its binding ability was not compromised. Interestingly, no Ca²⁺ ions were detected in the conserved calcium-binding site, although the module was properly folded; this suggests that the structural role of Ca²⁺ is less important than originally supposed. These observations indicate that despite their conserved function the scaffoldin-borne CBMs are more diverse in their sequences and structures than previously assumed.