Journal Article

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

Ali, Muhammad, Andersson, Eva, Jemth, Per, Benz, Caroline, Kliche, Johanna, Sayadi, Ahmed, Simonetti, Leandro, Davey, Norman E, Ivarsson, Ylva, Mihalic, Filip, Krystkowiak, Izabella

2022

Overview

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. Synopsis An optimized phage peptidome that tiles the disordered regions of the human proteome is presented, allowing the field of motif‐based interactions to transition into high‐throughput. Guidelines and tools for data analysis are provided. An optimized second generation human disorderome (HD2) phage library tiles all disordered regions from the human proteome. Different peptide display parameters are tested, including display on the major or minor coat proteins of the M13 phage, and splitting the library design based sub‐cellular localization of the peptide containing proteins. PepTools is a dedicated toolkit to annotate peptides and to identify consensus motifs. > 2,000 motif‐based interactions are presented, together with information on potential disease mutations or phosphorylation sites that might affect the interactions. Graphical Abstract An optimized phage peptidome that tiles the disordered regions of the human proteome is presented, allowing the field of motif‐based interactions to transition into high‐throughput. Guidelines and tools for data analysis are provided.

• Publisher: Nature Publishing Group UK,EMBO Press,John Wiley and Sons Inc,Springer Nature
• Subject: Amino acids / Binding Sites / Datasets / Design / EMBO22 / EMBO56 / Humans / intrinsically disordered regions / Libraries / Ligands / Localization / Mutation / Peptide Library / Peptides / Peptides - genetics / Peptides - metabolism / Phage display / Phages / Phosphorylation / Protein Binding / Protein interaction / protein-protein interactions / Proteins / Proteome - genetics / Proteome - metabolism / Proteomes / Proteomics / short linear motifs