Family 6 Carbohydrate Binding Modules Recognize the Non-reducing End of β-1,3-Linked Glucans by Presenting a Unique Ligand Binding Surface

Abstract

Enzymes that hydrolyze insoluble complex polysaccharide structures contain non-catalytic carbohydrate binding modules (CBMS) that play a pivotal role in the action of these enzymes against recalcitrant substrates. Family 6 CBMs (CBM6s) are distinct from other CBM families in that these protein modules contain multiple distinct ligand binding sites, a feature that makes CBM6s particularly appropriate receptors for the ␤-1,3-glucan laminarin, which displays an extended U-shaped conformation. To investigate the mechanism by which family 6 CBMs recognize laminarin, we report the biochemical and structural properties of a CBM6 (designated BhCBM6) that is located in an enzyme, which is shown, in this work, to display ␤-1,3-glucanase activity. BhCBM6 binds ␤-1,3-glucooligosaccharides with affinities of ϳ1 ؋ 10 5 M ؊1 . The x-ray crystal structure of this CBM in complex with laminarihexaose reveals similarity with the structures of other CBM6s but a unique binding mode. The binding cleft in this protein is sealed at one end, which prevents binding of linear polysaccharides such as cellulose, and the orientation of the sugar at this site prevents glycone extension of the ligand and thus conferring specificity for the non-reducing ends of glycans. The high affinity for extended ␤-1,3-glucooligosaccharides is conferred by interactions with the surface of the protein located between the two binding sites common to CBM6s and thus reveals a third ligand binding site in family 6 CBMs. This study therefore demonstrates how the multiple binding clefts and highly unusual protein surface of family 6 CBMs confers the extensive range of specificities displayed by this protein family. This is in sharp contrast to other families of CBMs where variation in specificity between different members reflects differences in the topology of a single binding site.

Recognition of the Helical Structure of β-1,4-Galactan by a New Family of Carbohydrate-binding Modules

“…This structural convergence of a carbohydrate binding motif is also quite remarkable considering that amylose and ␤-1,4-galactan possess helical structures of differing handedness and suggests that the aromatic platform is tailored more to accommodating the "tubelike" shape of the helices rather than the recognition of specific chemical groups. Consistent with this concept TmCBM41, an ␣-1,4-glucan binding CBM, did show some cross-specificity for pectic galactan (47). TmCBM61, however, showed no affinity for fragments of amylose, indicating that a polysaccharide having a helical structure with appropriate gross dimensions is a necessary condition for binding but not completely sufficient for a polysaccharide to act as a ligand for a CBM.…”

Domain Analysis of a Modular α- l -Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium Fibrobacter succinogenes S85

“…It is noteworthy that on the phylogenetic tree, although XynB3, SXA, and FSUAXH1 were members of cluster A, XynB3 and SXA clustered on the same branch while FSUAXH1 was located on a different branch. Structural and thermodynamic studies on CBM6 (1,6,11,18,44,62,63) have shown that this module by itself is functionally active. It would be interesting to compare the biochemistry of a cluster A GH43 fused directly at the C terminus to a CBM6 with a typical cluster A GH43 polypeptide (GH43 module/XX domain).…”

Substrate binding by a bacterial ABC transporter involved in polysaccharide export

“…7 were scored for conservation by using ConSurf (43,44) ars of xylans and cellulose (14,15). Similarily, the CBMs of laminarinase from Bacillus halodurans, and ␤-agarase from Saccharophagus degradans, specifically recognize the nonreducing terminus of their carbohydrate substrates (16,17). The current classification in the CAZy database does not include transport proteins.…”