The structural basis for the ligand specificity of family 2 carbohydrate-binding modules

The interactions of proteins with polysaccharides play a key role in the mi crobial hydrolysis of cellulose and xylan, the most abundant organic molecu les in the biosphere, and are thus pivotal to the recycling of photosynthet ically fixed carbon. Enzymes that attack these recalcitrant polymers have a modular structure comprising catalytic modules and non-catalytic carbohydr ate-binding modules (CBMs). The largest prokaryotic CBM: family, CBM2, cont ains members that bind cellulose (CBM2a) and xylan (CEM2b), respectively. A possible explanation for the different ligand specificity of CBM2b is that one of the surface tryptophans involved in the protein-carbohydrate intera ction is rotated by 90 degrees compared with its position in CBM2a (thus ma tching the structure of the binding site to the helical secondary structure of xylan), which may be promoted by a single amino acid difference between the two families. Here we show that by mutation of this single residue (Ar g-262-->Gly), a CBM2b xylan-binding module completely loses its affinity fo r xylan and becomes a cellulose-binding module. The structural effect of th e mutation has been revealed using NMR spectroscopy, which confirms that Tr p-259 rotates 90 degrees to lie flat against the protein surface. Except fo r this one residue, the mutation only results in minor changes to the struc ture. The mutated protein interacts with cellulose using the same residues that the wild-type CBM2b uses to interact with xylan, suggesting that the r ecognition is of the secondary structure of the polysaccharide rather than any specific recognition of the absence or presence of functional groups.