Journal Article

Proteome‐wide analysis of phospho‐regulated PDZ domain interactions

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

2018

Overview

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. Synopsis The study presents phosphomimetic proteomic peptide phage display, a novel method for exploring phospho‐regulated motif‐based interactions. Application to PDZ domains reveals a site‐specific phospho‐regulation of PDZ‐mediated interactions as a switching mechanism of interaction selectivity. Phosphomimetic proteomic peptide‐phage display (ProP‐PD) is a novel method for simultaneously finding motif‐based interaction and identifying phosphorylation switches. Site‐specific Ser/Thr phosphorylation events enable or disable PDZ domain interactions as revealed by phosphomimetic ProP‐PD. The approach can be used to explore potential phospho‐regulation of motif‐based interactions on a large scale. Graphical Abstract The study presents phosphomimetic proteomic peptide phage display, a novel method for exploring phospho‐regulated motif‐based interactions. Application to PDZ domains reveals a site‐specific phospho‐regulation of PDZ‐mediated interactions as a switching mechanism of interaction selectivity.

• Publisher: Nature Publishing Group UK,EMBO Press,John Wiley and Sons Inc,Springer Nature
• Subject: Amino Acid Sequence - genetics / Amino acids / Binders / Binding Sites / Biochemistry / Biokemi / Calorimetry / Disks Large Homolog 4 Protein - genetics / EMBO22 / EMBO31 / Humans / Ligands / Method / Methods / NMR / Nuclear magnetic resonance / Oligonucleotides / Oligonucleotides - genetics / PDZ domain / PDZ domainphage displayphosphorylationprotein–protein interactionScribble / PDZ Domains - genetics / Peptide Library / Peptides / Peptides - genetics / Phage display / Phages / Phosphorylation / Protein Binding - genetics / Protein interaction / Protein Interaction Mapping / Protein Interaction Maps - genetics / Proteins / protein–protein interaction / Proteome - genetics / Proteomes / Scribble / Switches / Switching / Thermophoresis / Titration / Titration calorimetry / Zonula Occludens-1 Protein - genetics