Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides

• Published in: International journal of biological macromolecules Vol. 120; no. Pt B; pp. 2509 - 2516
• Main Authors: Furtado, Gilvan Pessoa, Lourenzoni, Marcos Roberto, Fuzo, Carlos Alessandro, Fonseca-Maldonado, Raquel, Guazzaroni, María-Eugenia, Ribeiro, Lucas Ferreira, Ward, Richard J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-12-2018
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Firmicutes; Isothermal titration calorimetry; Molecular Dynamics Simulation; Molecular dynamics simulations; Mutation; Phage display; Polysaccharides - chemistry; Polysaccharides - metabolism; Protein Binding; Protein Domains; Protein Engineering; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Phage display; AE; epPCR; ITC; Isothermal titration calorimetry; Molecular dynamics simulations; RtCBM11; RtCBM44; XegA; CBM
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2018.09.022

Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide β-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage library created by error prone PCR for xyloglucan binding identified RtCBM11 mutants with enhanced xyloglucan affinity. Subsequent recombination of the selected variants by site-directed mutagenesis generated the H102L/Y152F and Y46N/G52D/H102L/Y152F mutants. Fusion of the quadruple RtCBM11 mutant with the xyloglucanase from Aspergillus niveus increased the catalytic efficiency of the enzyme by 38%. Isothermal titration calorimetry demonstrated increased xyloglucan affinity for both mutants and reduced affinity for β-glucan in the H102L/Y152F mutant. Molecular dynamics simulations indicated that the increased xyloglucan specificity results both from formation of a xylosyl binding pocket in the carbohydrate binding cleft, and via modulation of a hydrogen bond network between the oligosaccharide ligand and the protein. These results explain the improved xyloglucan binding in the RtCBM11 H102L/Y152F mutant and advance the understanding of the structural determinants of CBMs binding that discriminate between branched and unbranched polysaccharides.