Discovery of short linear motif‐mediated interactions through phage display of intrinsically disordered regions of the human proteome. (18th January 2017)

• Record Type: Journal Article
• Authors: Davey, Norman E.; Seo, Moon‐Hyeong; Yadav, Vikash Kumar; Jeon, Jouhyun; Nim, Satra; Krystkowiak, Izabella; Blikstad, Cecilia; Dong, Debbie; Markova, Natalia; Kim, Philip M.; Ivarsson, Ylva
• Is Part Of: FEBS journal. Volume 284:Number 3(2017)
• Page Start: 485
• Page End: 498
• Publication Date: 2017-01-18
• Subjects: EVH1 domain -- PDZ domain -- Protein–protein interactions -- short linear motifs -- VHS domain; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.13995
• Languages: English
• ISSNs: 1742-464X

Abstract

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. Abstract : The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs. These compact interfaces provide binding sites for peptide‐binding domains. We present a novel proteomic peptide phage display library that displays peptides directly mapping to disordered regions of the human proteome and validate its performance. This opens for large‐scale analysis of motif‐based interactions.