Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

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, GM 1 pentasaccharides (β-Gal-, 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.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“… 362 − 371 Other notable applications are the characterization of ligands with weak affinity such as metals 299 , 371 − 376 or carbohydrates. 285 , 377 For proteins with multiple distinct ligand binding sites, it has been shown that by performing a concentration series it is possible to characterize individual interactions. 264 , 378 The strength of HDX-MS to provide detailed insight into such a wide range of interactions continues to drive its use to study increasingly complex biological systems.…”

“…To best resolve differences upon binding, it is important to make the comparison between a fully free population and an as-fully bound-as-possible population. With weak binding interactions it is often difficult to achieve saturation, as it may require very high concentrations of ligand. , Often even a several-fold molar excess of ligand is not sufficient for achieving >99% of the protein bound in solution. Furthermore, a complex should be formed prior to deuterium exchange to ensure the fully bound state is being probed.…”

“…Beyond epitope mapping, HDX-MS continues to be utilized for studying protein–protein interactions in complex protein systems and revealing mechanisms of activation in a range of systems including: clotting factors, ,− metabolic enzymes, ,,− heat shock proteins ,, and other chaperones, − signaling complexes, − and complexes exclusively residing in membranes . HDX-MS has also been extensively used to study interactions between proteins with nucleic acids, including activation of transcription factors. − Other notable applications are the characterization of ligands with weak affinity such as metals ,− or carbohydrates. , For proteins with multiple distinct ligand binding sites, it has been shown that by performing a concentration series it is possible to characterize individual interactions. , The strength of HDX-MS to provide detailed insight into such a wide range of interactions continues to drive its use to study increasingly complex biological systems.…”

Structure-Activity Relationships of Baicalein and its Analogs as Novel TSLP Inhibitors

“…The binding site of 1 in hTSLP was confirmed using hydrogen-deuterium exchange (HDX)-MS. HDX-MS monitors the exchange between deuterium in the solvent and backbone amide hydrogen, which generally provides information on the binding of a compound to a protein 24,25 . Following the addition of 1 , the N -terminal half of the H1 helix in hTSLP showed decreased deuterium uptake as illustrated in Fig.…”

Combining Mass Spectrometry, Surface Acoustic Wave Interaction Analysis, and Cell Viability Assays for Characterization of Shiga Toxin Subtypes of Pathogenic Escherichia coli Bacteria

“…Sophisticated ESI MS technologies have been described allowing for determination of the stoichiometry of multivalent complexes of Stx with Gb3 and the affinities of Stx1 and Stx2 to Gb3 analogs, , localization of the carbohydrate binding sites of the B-subunit polymer of Stx1a, detection of specific interactions between Gb3Cer and Stx, and investigations on the assembly and stability of Stx . Safe and effective means of detecting and quantitating various Stx subtypes have been recently reported by Silva et al They were able to distinguish among most of the known subtypes including Stx1a, Stx2a, and Stx2e.…”