Unravelling carbohydrate binding module 21 (CBM21) dynamics of interaction with amylose

The carbohydrate binding module 21 (CBM21) from Rhizopus oryzae is a dual-site CBM proposed to disrupt polysaccharide structures. Additionally, it serves as a purification tag in industry. CBM21 crystal structure features a Glc residue in an unusual ¹S₃ conformation, whose relevance for the CBM mechanism of action is unclear. In this context, we seek to contribute for the understanding of CBM21 mechanism of action by: i) investigating the role of the ¹S₃ conformation on carbohydrate recognition, and ii) characterize the protein-carbohydrate binding dynamics using molecular dynamics and metadynamics simulations at MM and QM/MM levels. Results indicate the ¹S₃ Glc conformation is unlikely to occur under biological conditions, being originated from the crystallographic environment. CBM21 binding to small ligands appears transient and unstable, while protein dimerization and polysaccharide chain size influence complex stability. In interactions with amylose, CBM21 exhibits a repeated unbinding followed by re-binding, while simultaneously alternating between binding sites I and II. These results suggest that CBM21 acts through transient interactions, directing carbohydrates to the catalytic center rather than forming strong and long-lasting bonds with carbohydrates. Accordingly, we expect such atomistic depiction of CBM21 mechanism could aid in CBM design targeting biotechnological applications.