Proteome-wide analysis of phospho-regulated PDZ domain interactions

Gustav N. Sundell, Roland Arnold*, Muhammad Ali, Piangfan Naksukpaiboon, Julien Orts, Peter Güntert, Celestine N. Chi, Ylva Ivarsson

*Corresponding author for this work

Research output: Contribution to journalArticle

14 Citations (Scopus)
15 Downloads (Pure)

Abstract

A key function of reversible protein phosphorylation is to regulate protein–protein interactions, many of which involve short linear motifs (3–12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale.

Original languageEnglish
Article numbere8129
Pages (from-to)1-22
JournalMolecular systems biology
Volume14
Issue number8
DOIs
Publication statusPublished - 01 Aug 2018
Externally publishedYes

Keywords

  • PDZ domain
  • phage display
  • phosphorylation
  • protein–protein interaction
  • Scribble

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Agricultural and Biological Sciences(all)
  • Applied Mathematics

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