Proteome-wide analysis of phospho-regulated PDZ domain interactions

Mol Syst Biol. 2018 Aug 20;14(8):e8129. doi: 10.15252/msb.20178129.


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.

Keywords: PDZ domain; Scribble; phage display; phosphorylation; protein–protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence / genetics
  • Binding Sites
  • Disks Large Homolog 4 Protein / genetics
  • Humans
  • Ligands
  • Oligonucleotides / genetics
  • PDZ Domains / genetics*
  • Peptide Library
  • Peptides / genetics*
  • Phosphorylation
  • Protein Binding / genetics
  • Protein Interaction Mapping
  • Protein Interaction Maps / genetics*
  • Proteome / genetics*
  • Zonula Occludens-1 Protein / genetics


  • Disks Large Homolog 4 Protein
  • Ligands
  • Oligonucleotides
  • Peptide Library
  • Peptides
  • Proteome
  • TJP1 protein, human
  • Zonula Occludens-1 Protein

Associated data