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Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome
Univ Coll Dublin, Conway Inst Biomol & Biomed Sci, Dublin 4, Ireland..
Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
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2017 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 3, p. 485-498Article in journal (Refereed) Published
Abstract [en]

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 mu M 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.

Place, publisher, year, edition, pages
2017. Vol. 284, no 3, p. 485-498
Keywords [en]
EVH1 domain, PDZ domain, Protein-protein interactions, short linear motifs, VHS domain
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-317956DOI: 10.1111/febs.13995ISI: 000393601800011PubMedID: 28002650OAI: oai:DiVA.org:uu-317956DiVA, id: diva2:1086267
Available from: 2017-03-31 Created: 2017-03-31 Last updated: 2017-11-29Bibliographically approved

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