title: Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry.
abstract: Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.
authors.affiliation: Department of Pharmacy, University of Copenhagen , Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Leurs
authors.forename: Brian
authors.initials: B
authors.lastname: Lohse
authors.forename: Shonoi
authors.initials: S
authors.lastname: Ming
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
authors.forename: Rasmus P
authors.initials: RP
authors.lastname: Clausen
authors.forename: Jesper L
authors.initials: JL
authors.lastname: Kristensen
authors.forename: Kasper D
authors.initials: KD
authors.lastname: Rand
mesh_terms: D001665:Binding Sites; D041961:Deuterium Exchange Measurement; D056466:Histone Demethylases / Q000737:chemistry*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases / Q000737:chemistry*; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*
chemical_list: C413655:KDM4C protein, human; D008024:Ligands; D010455:Peptides; D056466:Histone Demethylases; D056484:Jumonji Domain-Containing Histone Demethylases; C495195:KDM1A protein, human