Ligand-mediated dimerization of a carbohydrate-binding module reveals a novel mechanism for protein-carbohydrate recognition

Flint, James and Nurizzo, Didier and Harding, Stephen E. and Longman, Emma and Davies, Gideon J. and Gilbert, Harry J. and Bolam, David N. (2004) Ligand-mediated dimerization of a carbohydrate-binding module reveals a novel mechanism for protein-carbohydrate recognition. Journal of Molecular Biology, 337 (2). pp. 417-426. ISSN 0022-2836

Full content URL: http://dx.doi.org/10.1016/j.jmb.2003.12.081

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Item Type:Article
Item Status:Live Archive

Abstract

The structural and thermodynamic basis for carbohydrate-protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two "family 29" carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the β-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this "dimeric" binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein modules, leading to ligand-induced formation of the binding site, represents a completely novel mechanism for protein-carbohydrate recognition that may mimic that displayed by naturally dimeric protein-carbohydrate interactions. © 2004 Elsevier Ltd. All rights reserved.

Keywords:carbohydrate binding protein, carbohydrate derivative, cellohexaose, cellulose, mannan, mannohexaose, mutant protein, non catalytic protein 1, oligosaccharide, protein, unclassified drug, xylan, article, binding site, calorimetry, complex formation, controlled study, dimerization, molecular interaction, molecular mechanics, molecular recognition, nonhuman, physical chemistry, priority journal, protein binding, protein domain, protein structure, stoichiometry, ultracentrifugation, Base Sequence, Binding Sites, Carbohydrate Metabolism, Carbohydrates, Cellulase, Crystallography, X-Ray, DNA, Fungal, Fungal Proteins, Glycoside Hydrolases, Ligands, Macromolecular Substances, Models, Molecular, Multienzyme Complexes, Mutagenesis, Site-Directed, Oligosaccharides, Piromyces, Thermodynamics, Ceratonia siliqua, Piromyces equi
Subjects:C Biological Sciences > C720 Biological Chemistry
Divisions:College of Science > School of Life Sciences
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ID Code:12474
Deposited On:15 Dec 2013 20:40

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