title: Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome.
abstract: The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 μm through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.
authors.affiliation: Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Ireland.
authors.forename: Norman E
authors.initials: NE
authors.lastname: Davey
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada.
authors.forename: Moon-Hyeong
authors.initials: MH
authors.lastname: Seo
authors.affiliation: Department of Chemistry - BMC, Uppsala University, Sweden.
authors.forename: Vikash Kumar
authors.initials: VK
authors.lastname: Yadav
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada.
authors.forename: Jouhyun
authors.initials: J
authors.lastname: Jeon
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada.
authors.forename: Satra
authors.initials: S
authors.lastname: Nim
authors.affiliation: Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Ireland.
authors.forename: Izabella
authors.initials: I
authors.lastname: Krystkowiak
authors.affiliation: Department of Chemistry - BMC, Uppsala University, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Blikstad
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada.
authors.forename: Debbie
authors.initials: D
authors.lastname: Dong
authors.affiliation: Malvern Instruments Nordic AB, Solna, Sweden.
authors.forename: Natalia
authors.initials: N
authors.lastname: Markova
authors.affiliation: Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada.|Department of Molecular Genetics and Department of Computer Science, University of Toronto, Canada.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Kim
authors.affiliation: Department of Chemistry - BMC, Uppsala University, Sweden.
authors.forename: Ylva
authors.initials: Y
authors.lastname: Ivarsson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D020543:Proteome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D064267:Intrinsically Disordered Proteins; D019151:Peptide Library; D010455:Peptides; D020543:Proteome; D011994:Recombinant Proteins
keywords: EVH1 domain; PDZ domain; Protein-protein interactions; VHS domain; short linear motifs