Ribeiro, DO; Viegas, A; Pires, VMR; Silva, JM-; Bule, P; Chai, W; Marcelo, F; Fontes, CMGA; Cabrita, EJ; Palma, AS; Carvalho, AL
Understanding the specific molecular interactions between proteins and β1,3- 1,4-mixed-linked D-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognise them. The family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic bio-mass, 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 β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the b1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc 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 β1,3-1,4-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 β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain.
"...the structural details here revealed on the CtCBM11 ligand recognition site may influence the planning and development of efficient and low-cost mechanisms for the conversion of biomass into usable sources of energy, as well as into nutrients for animal feedstock."
In the context of the cellulosome, the structural details here revealed on the CtCBM11 ligand recognition site may influence the planning and development of efficient and low-cost mechanisms for the conversion of biomass into usable sources of energy, as well as into nutrients for animal feedstock. Additionally, the understanding, at the molecular level, of how CtCBM11 selects and binds its ligands may inspire the design of new biomolecules with improved capabilities to be explored in health and agriculture applications.
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