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

Abstract

The microbial enzymes that depolymerize plant cell wall polysaccharides, ultimately promoting energy liberation and carbon recycling, are typically complex in their modularity and often contain carbohydrate-binding modules (CBMs). Here, through analysis of an unknown module from a Thermotoga maritima endo-␤-1,4-galactanase, we identify a new family of CBMs that are most frequently found appended to proteins with ␤-1,4-galactanase activity. Polysaccharide microarray screening, immunofluorescence microscopy, and biochemical analysis of the isolated module demonstrate the specificity of the module, here called TmCBM61, for ␤-1,4-linked galactose-containing ligands, making it the founding member of family CBM61. The ultra-high resolution x-ray crystal structures of TmCBM61 (0.95 and 1.4 Å resolution) in complex with ␤-1,4-galactotriose reveal the molecular basis of the specificity of the CBM for ␤-1,4-galactan. Analysis of these structures provides insight into the recognition of an unexpected helical galactan conformation through a mode of binding that resembles the recognition of starch.

Function of bacteriophage G7C esterase tailspike in host cell adsorption

“…Notably, almost all DALI hits, including the most similar ones, show sequence identity to gp63.1 below 10% with only a few at 12–15%. Nevertheless, the substrate‐binding site in several best structural matches [PDB code 2XON (Cid et al ., ) and 2W87 (Montanier et al ., )] roughly overlaps with a shallow cavity of gp63.1 that binds a di‐ethylene glycol molecule in an extended conformation (this compound is likely to be a result of decomposition of polyethylene glycol used in crystallization). The di‐ethylene glycol is likely to mimic an extended polysaccharide chain.…”

Structure of the full‐length insecticidal protein Cry1Ac reveals intriguing details of toxin packaging into in vivo formed crystals

“…(A)]. Domains V and VII are beta‐rolls (similar to D‐II or D‐III) that closely resemble carbohydrate‐binding modules (CBM) found in sugar hydrolases (2ZEW, 2XON), however, it is difficult to guess which particular carbohydrates (if any) may serve as their ligands because residues on the putative sugar‐binding interfaces are conserved neither in sequence nor in local structure [Fig. (B)].…”

Advances in molecular engineering of carbohydrate-binding modules

“…For CBM61 from the Thermotoga maritima endo‐β‐1,4‐galactanase, the W594A mutation abolishes binding to β‐1,4‐galactan, and mutation of either tryptophan, W508A or W563A, results in a 10‐fold reduction in affinity. These amino acids are also important for adopting the curvature of the ligand, giving them a critical role in galactan binding …”