Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium thermocellum

Understanding the specific molecular interactions between proteins and beta 1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for beta 1,3-1,4-mixed-linked over beta 1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to beta 1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the beta 1,3-linkage are important for recognition, and identified the tetrasaccharide Glc beta 1,4Glc beta 1,4Glc beta 1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of beta 1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-pi stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a beta 1,3-linkage at the reducing end and at specific positions along the beta 1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked beta-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. Database Structural data are available in the Protein Data Bank under the accession codes and .