Cello-oligomer-binding dynamics and directionality in family 4 carbohydrate-binding modules

Carbohydrate-binding modules (CBMs) play significant roles in modulating the function of cellulases, and understanding the protein-carbohydrate recognition mechanisms by which CBMs selectively bind substrate is critical to development of enhanced biomass conversion technology. CBMs exhibit a limited range of specificity and appear to bind polysaccharides in a directional fashion dictated by the position of the ring oxygen relative to the protein fold. The two family 4 CBMs of Cellulomonas fimi Cel9B (CfCBM4) are reported to preferentially bind cellulosic substrates. However, experimental evidence suggests that these CBMs may not exhibit a thermodynamic preference for a particular orientation. We use molecular dynamics (MD) and free energy calculations to investigate protein-carbohydrate recognition mechanisms in CfCBM4-1 and CfCBM4-2 and to elucidate preferential ligand-binding orientation. We evaluate four cellopentaose orientations including that of the crystal structure and three others suggested by nuclear magnetic resonance (NMR). These four orientations differ based on position of the ligand reducing end (RE) and pyranose ring orientations relative to the protein core. MD simulations indicate that the plausible orientations reduce to two conformations. Calculated ligand-binding free energy discerns each of the orientations is equally favorable. The calculated free energies are in excellent agreement with isothermal titration calorimetry measurements from the literature. MD simulations further reveal the approximate structural symmetry of the oligosaccharides relative to the amino acids along the binding cleft plays a role in the promiscuity of ligand binding. A survey of ligand-bound structures suggests this phenomenon may be characteristic of the broader class of proteins belonging to the β-sandwich fold.