title: Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry.
abstract: The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.|Alberta Glycomics Centre, Edmonton, Alberta, Canada.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.|Alberta Glycomics Centre, Edmonton, Alberta, Canada.
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.|Alberta Glycomics Centre, Edmonton, Alberta, Canada.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.|Alberta Glycomics Centre, Edmonton, Alberta, Canada.
authors.forename: Luiz
authors.initials: L
authors.lastname: Eugenio
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.|Alberta Glycomics Centre, Edmonton, Alberta, Canada.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Ng
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada. john.klassen@ualberta.ca.|Alberta Glycomics Centre, Edmonton, Alberta, Canada. john.klassen@ualberta.ca.
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry*; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006860:Hydrogen Bonding; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002241:Carbohydrates; D011506:Proteins
keywords: Hydrogen bonds; Hydrogen/deuterium exchange mass spectrometry; Protein–carbohydrate complexes