Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome

作者：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

来源：FEBS Journal, 2017, 284(3): 485-498.

DOI：10.1111/febs.13995

• EVH1 domain
• PDZ domain
• Protein-protein interactions
• short linear motifs
• VHS domain

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.

• 出版日期2017-2