Proteome‐scale mapping of binding sites in the unstructured regions of the human proteome

Abstract

Specific protein–protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein–protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type‐specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide‐phage display library that tiles all disordered regions of the human proteome and allows the screening of ~ 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. Finally, we show how the amino acid resolution‐binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome‐wide discovery of SLiM‐based interactions.

Sequence- and chemical specificity define the functional landscape of intrinsically disordered regions

“…Generally, these two modes of interaction are discussed separately. Motifs are considered in the context of specific stoichiometric interactions, while distributed multivalent binding is mostly associated with biomolecular condensates ( 10, 16, 18, 22 ). However, our results prompted us to wonder if these two interaction modes might instead exist on a combined two-dimensional landscape ( Fig.…”

“…Despite their importance for function, IDRs are often poorly conserved as assessed by alignment-based methods (4)(5)(6)(7). The notable exceptions to this are short linear motifs (SLiMs), conserved stretches of 5 to 15 amino acids that define sequence-specific recognition sites (8)(9)(10). The modular nature of many SLiMs is demonstrated by their ability to mediate molecular recognition when inserted into otherwise neutral contexts (10,11).…”

“…The notable exceptions to this are short linear motifs (SLiMs), conserved stretches of 5 to 15 amino acids that define sequence-specific recognition sites (8)(9)(10). The modular nature of many SLiMs is demonstrated by their ability to mediate molecular recognition when inserted into otherwise neutral contexts (10,11). In addition to molecular recognition driven by SLiMs, IDRs that lack SLiMs can engage in distributed multivalent interactions driven by sequence-encoded chemistry (3,(12)(13)(14)(15).…”

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

“…Of 220 viral SLiMs from the benchmarking set that were present in the RiboVD library, 53 were re-discovered by the selections (Figure 1C; Table S4). The motif -rediscovery rate (24% recall) was high, surpassing our recent benchmarking results against a human disorderome phage library (19.3% rediscovery) (Benz et al, 2022). We further compiled a virus-host PPI reference set based on data available in IntAct (Orchard et al, 2014), BioGrid (Oughtred et al, 2021), VirHostNet (Guirimand et al, 2015) and other published sources (Table S5).…”

“…Additionally, more than 200,000 virus-host PPIs have been suggested from computational structure-based pan-viral analyses (Lasso et al, 2019). However, SLiM-based interactions are likely underrepresented in the available large-scale virus-host PPI datasets because the methods used are not optimized to capture low-affinity transient SLiM-based interactions (Benz et al, 2022; Cluet et al, 2020). Consequently, most SLiM-based virus-host PPIs have been identified using low-throughput methods (Kumar et al, 2022).…”