CBM21 uses a mobility-dependent mechanism to interact with amylose fibers

Carbohydrate-binding modules (CBMs) are a family of sugar-binding proteins that enhance the catalytic properties of associated enzymes. Among these, CBM21 specializes in starch recognition and is used as a protein tag in industrial applications. CBM21 exhibits low binding affinity toward glycans and frequently unbinds and rebinds to amylose through two distinct binding sites. Using multiple microsecond, we analyzed CBM21 interactions with long amylose chains. CBM21 was observed to move unidirectionally along amylose fibers toward their non-reducing ends, which are the preferred catalytic sites for glycoside hydrolase 15 (GH15), to which CBM21 is linked. This motion is driven by the entropy of the amylose fiber. In contrast, CBM3a, a protein that translocates along cellulose on the second timescale, showed no significant movement in similar simulations, possibly due to its higher dissociation free energy (90.8 kJ/mol for CBM3a vs. 30.3 kJ/mol for CBM21). Markov State Models suggested a potential third CBM21 binding site formed by Tyr14, Tyr16, and Tyr102, topologically opposed to site 1. Aromatic residues on the protein surface may guide its motion along amylose. This study provides novel insights into CBMs interactions, movement, and functional mechanisms, offering a basis for new biotechnological applications.