(304b) Carbohydrate Recognition Mechanisms in Cellulose-Specific Type B Carbohydrate Binding Modules

Effective enzymatic degradation of crystalline polysaccharides requires a synergistic cocktail of hydrolytic enzymes tailored to the wide-ranging degrees of substrate crystallinity. To accomplish this type of targeted carbohydrate recognition, nature produces multi-modular enzymes, having at least one catalytic domain appended to one or more carbohydrate binding modules (CBM). Type B CBMs encompass several families (i.e., protein folds) of CBMs that are generally thought to selectively bind oligomeric polysaccharides; however, a subset of cellulose-specific CBM families (4, 17, and 28) appear to bind non-crystalline cellulose more tightly than oligomers and in a manner that discriminates between surface topology. In this study, we investigated the molecular-level origins of oligomeric and non-crystalline carbohydrate recognition in cellulose-specific Type B CBMs using molecular dynamics simulation and free energy calculations. Examining two CBMs from each of the three families, we describe how protein-ligand dynamics contribute to observed variations in oligomeric binding affinity within the same CBM family. Comparisons across the three CBM families identified factors leading to modified functionality prohibiting competitive binding, despite sequential and specificity similarities. Using Free Energy Perturbation with Hamiltonian Replica Exchange Molecular Dynamics, we examined the hypothesis that the open topology of the binding grooves in families 17 and 28 necessitate tight binding of an oligomer, while the more confined family 4 binding groove does not require the same degree of tight binding. Finally, we elucidated the mechanisms of non-crystalline carbohydrate recognition by modeling a family 28 CBM complexed with a partially descrystallized cellulose substrate. Molecular simulation provided structural and dynamic data for direct comparison to oligomeric modes of carbohydrate recognition, and thermodynamic integration was used to determine ligand binding free energy. Comparing both protein-carbohydrate interactions and ligand binding free energies, which were within error of experimental values, we validated the correlation of high- and low-affinity binding sites with non-crystalline and oligomeric binding, respectively. Our study provides an unprecedented level of insight into the complex solid and soluble carbohydrate substrate recognition mechanisms of CBMs, the findings of which hold considerable promise for enhancing lignocellulosic biomass conversion technology.