Functional and Structural Determination of a CBM101 Family Carbohydrate-Binding Module (CBM): A Single Residue Change Reshapes CBM Specificity

Carbohydrate-binding modules (CBMs) make up a class of protein domains that are favorable tools for the studies and applications of carbohydrates. Agarose and funoran are galactans derived from red algae with structural similarities and subtle differences. The CBM101 family was established following the discovery of WfCBM101, which showed a binding capacity to agarose. Herein, we performed functional and structural analyses of WfCBM101. The results showed that WfCBM101 binds to funoran, which represents the first identified funoran-binding CBM. The structure of WfCBM101 was resolved at a 1.5 Å resolution by X-ray crystallography. The protein displays a β-sandwich fold with two antiparallel β-sheets composed of 7 β-strands. Mutagenesis studies revealed an unusual phenomenon in which the mutation of a single residue K96 reshapes the protein specificity. These findings highlight the fine-tuned recognition and binding capacities of CBMs, and they may facilitate the future discovery of carbohydrate-binding proteins.