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

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 lM through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using fulllength proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PDderived, 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.

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

“…Nucleic acids research 40, 10628-10641. Davey, N.E., Seo, M.H., Yadav, V.K., Jeon, J., Nim, S., Krystkowiak, I., Blikstad, C., Dong, D., Markova, N., Kim, P.M., et al (2017). Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome.…”

Protein Serine/Threonine Phosphatases: Keys to Unlocking Regulators and Substrates

“…New bioinformatics tools, such as SLiMSearch (173) and Pro-PD phage display libraries (163), were instrumental in identifying the PP2A B56 SLiM. When utilized in conjunction with the continuously expanding information on the human phosphoproteome (27), these experimental approaches should accelerate the discovery of PP2A substrates and regulators (170,174,175).…”

High‐throughput discovery of functional disordered regions: investigation of transactivation domains

“…In vitro technologies such as phage display are powerful to identify short disordered linear motifs (three to seven residues within IDRs) that can mediate interactions with specific protein domains in vitro as well as discover strong binders (Ivarsson et al , ; Garrido‐Urbani et al , ; Davey et al , ). Such approaches require screening short peptides against specific interaction partners, thus constraining the mechanism by which they mediate function (Jones et al , ; Ivarsson et al , ; Garrido‐Urbani et al , ; Davey et al , ). The screening occurs outside of the relevant cellular/biological context during the selection experiment and hence does not explicitly consider cellular specificity for binding, i.e., selection against promiscuous binding with other molecules in the cell (negative selection).…”