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Discovery of short linear motif‐mediated interactions through phage display of intrinsically disordered regions of the human proteome
Author(s) -
Davey Norman E.,
Seo MoonHyeong,
Yadav Vikash Kumar,
Jeon Jouhyun,
Nim Satra,
Krystkowiak Izabella,
Blikstad Cecilia,
Dong Debbie,
Markova Natalia,
Kim Philip M.,
Ivarsson Ylva
Publication year - 2017
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13995
Subject(s) - proteome , isothermal titration calorimetry , phage display , computational biology , human proteome project , peptide library , protein–protein interaction , biology , binding selectivity , peptide , plasma protein binding , intrinsically disordered proteins , binding site , structural motif , sequence motif , proteomics , biochemistry , peptide sequence , dna , gene
The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs ( SL iMs), 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 SL iMs 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 SL iMs in the proteome. The performance of the ProP‐ PD library was validated through selections against SL iM‐binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SL iM‐binding specificities of the bait proteins. For SHANK 1 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 GGA 1 and three identified ligands to 40–130 μ 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, SL iM‐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.