Proteome‐scale amino‐acid resolution footprinting of protein‐binding sites in the intrinsically disordered regions

Abstract

Protein‐protein interactions are central to cell function. Numerous studies have provided large‐scale information on human protein‐protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type‐specific interactions disproportionately under‐represented. I will present our efforts towards finding the short linear motif (SLiM)‐based interactions of a variety of protein domains. I will present an optimized proteomic peptide‐phage display (ProP‐PD) 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, and the tools and guidelines we have defined for processing the data (1). Using the approach we identified >2,000 interaction pairs for 35 known SLiM‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. The amino acid resolution binding site information can be used to pin‐point functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. Finally, I will describe how we developed the approach for large‐scale discovery of coronavirus‐host factor protein interactions (2). We screened more than 130 protein domains for binding to viral peptides, and translated the high‐resolution information on direct virus‐host interactions to a specific peptide‐based antiviral inhibitor of an interaction between the G3BP1/2 proteins and an ΦxFG peptide motif in the SARS‐CoV‐2 nucleocapsid (N) protein. ProP‐PD may thus be used both to illuminate the motif‐based part of the interactome and to uncover leads for innovative inhibitor design.References Benz et al., (2022) Proteome‐scale mapping of binding sites in the unstructured regions of the human proteome. Mol Syst Biol. 2022 Jan;18(1):e10584. Kruse et al., (2021) Large scale discovery of coronavirus‐host factor protein interaction motifs reveals SARS‐CoV‐2 specific mechanisms and vulnerabilities. Nat Commun. 2021 Nov 19;12(1):6761.