Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome.
Journal article
Authors | Yadav, V. |
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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. |
Keywords | Short Linear Motifs ; PPI; ProP-PD; Binding peptides |
Year | 2016 |
Journal | The FEBS journal |
Journal citation | Vol 284 (Issue 3), pp. 485-498 |
Publisher | FEBS |
Wiley Online Library | |
ISSN | 1742-4658 |
1742-464X | |
Digital Object Identifier (DOI) | https://doi.org/10.1111/febs.13995 |
Web address (URL) | https://doi.org/10.1111/febs.13995 |
https://pubmed.ncbi.nlm.nih.gov/28002650/ | |
Output status | Published |
Publication dates | |
Online | 21 Dec 2016 |
18 Jan 2017 | |
Online | 07 Feb 2017 |
Publication process dates | |
Accepted | 19 Dec 2016 |
Deposited | 15 Jun 2023 |
https://repository.derby.ac.uk/item/9z4xv/discovery-of-short-linear-motif-mediated-interactions-through-phage-display-of-intrinsically-disordered-regions-of-the-human-proteome
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