A single mutation reforms the binding activity of an adhesion-deficient family 3 carbohydrate-binding module

The crystal structure of the family 3b carbohydrate-binding module (CBM3b) of the cellulosomal multimodular hydrolytic enzyme cellobiohydrolase 9A (Cbh9A) from Clostridium thermocellum has been determined. Cbh9A CBM3b crystallized in space group P4₁ with four molecules in the asymmetric unit and diffracted to a resolution of 2.20 Å using synchrotron radiation. The structure was determined by molecular replacement using C. thermocellum Cel9V CBM3b′ (PDB entry 2wnx) as a model. The C. thermocellum Cbh9A CBM3b molecule forms a nine-stranded antiparallel β-sandwich similar to other family 3 carbo­hydrate-binding modules (CBMs). It has a short planar array of two aromatic residues that are assumed to bind cellulose, yet it lacks the ability to bind cellulose. The molecule contains a shallow groove of unknown function that characterizes other family 3 CBMs with high sequence homology. In addition, it contains a calcium-binding site formed by a group of amino-acid residues that are highly conserved in similar structures. After determination of the three-dimensional structure of Cbh9A CBM3b, the site-specific N126W mutant was produced with the intention of enhancing the cellulose-binding ability of the CBM. Cbh9A CBM3b^N126W crystallized in space group P4₁2₁2, with one molecule in the asymmetric unit. The crystals diffracted to 1.04 Å resolution using synchrotron radiation. The structure of Cbh9A CBM3b^N126W revealed incorporation of the mutated Trp126 into the putative cellulose-binding strip of residues. Cellulose-binding experiments demonstrated the ability of Cbh9A CBM3b^N126W to bind cellulose owing to the mutation. This is the first report of the engineered conversion of a non-cellulose-binding CBM3 to a binding CBM3 by site-directed mutagenesis. The three-dimensional structure of Cbh9A CBM3b^N126W provided a structural correlation with cellulose-binding ability, revealing a longer planar array of definitive cellulose-binding residues.