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 40-130μm 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.
Highlights
Recent years have seen a tremendous growth in the number of characterized human protein–protein interactions (PPI)
Peptides were tiled with an overlap of seven amino acids between peptides to optimize the display of the peptides and the coverage of intact short linear motif (SLiM) (Fig. 1A)
The SLiM-based interactions are crucial for cell function but notoriously difficult to discover by most methods developed to explore PPIs on large scale
Summary
Recent years have seen a tremendous growth in the number of characterized human protein–protein interactions (PPI). A large-scale yeast-two-hybrid (Y2H) screen provided information on 14 000 potential binary PPI [1] and high-throughput affinity-purification coupled to mass spectrometry (AP-MS) of ~ 2600 bait proteins in HEK293T cells revealed more than 23 000 binary interactions or complexes [2]. In these datasets there are striking numbers of new interactions, bearing witness to the large set of unknown PPIs awaiting discovery. Due to their central role in cell physiology SLiM-mediated interactions drive evolution of signaling networks [6,7], are frequently deregulated in diseases such as cancers [8] and are often mimicked by pathogens to hijack host systems [9,10,11]
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