Proteome‐wide analysis of phospho‐regulated PDZ domain interactions

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.

Crystal structure of the human Scribble PDZ1 domain bound to the PDZ‐binding motif of APC

“…We now show that human Scribble binds the APC PBM with low micromolar affinities, with SCRIB PDZ1 domain being the highest affinity site with a K D of 6.0 μ m , whereas SCRIB PDZ3 binds with 18.3 μ m and the SCRIB PDZ2 an affinity of 36.0 μ m . These findings are in disagreement with previous data indicating that APC interacts with SCRIB PDZ1 and 4 domains or with SCRIB PDZ1 and 2 domain only . Instead, the pattern in interactions and affinities we observed for Scribble APC interactions resembles the binding pattern for SCRIB:β‐PIX , which also binds SCRIB PDZ domains 1,2 and 3, with PDZ1 being the highest affinity interactor and PDZ2 the weakest binder.…”

“…Importantly, knockdown of human Scribble by RNAi in Caco‐2 cells disrupted the proper localisation of APC at adherens junction . Intriguingly, APC was shown to engage Scribble through its C‐terminal PDZ binding motif binding specifically to the Scribble PDZ1 and 4 domains , however a recent report using peptide‐phage display proteomics approach indicates that the APC C‐terminal PDZ binding motif binds directly to Scribble PDZ1 and 2 .…”

PDZ Domains as Drug Targets

“…c) In ZO-1 PDZ2, the domain folds from two nonfunctional monomeric domains where βB and βC of the two domains swap in order to complete the fold into the functional dimeric PDZ tandem. [44,45] In addition to the carboxylate-dependent binding of PDZ domains, it has more recently been demonstrated that PDZ domains can also bind internal peptide motifs [42,[46][47][48][49] and phospholipids. e) PDZ domain ligands can also bind using internal motifs where it usually still relies on the insertion of a hydrophobic residue into the binding pocket and either hydrophobic or hydrogen bond interaction coordination of the P −2 , but the interaction also relies on the P +1 or P +2 residue being negatively charged (Glu/Asp), using the carboxylic acid as substitute for the C-terminal.…”

Specificity in PDZ-peptide interaction networks: Computational analysis and review

“…Specifically, proteomic analysis of binding interactions to either a phosphorylated or unphosphorylated version of the C-terminal peptide of ribosomal S6 kinase 1 (RSK1), which can be phosphorylated in response to epidermal growth factor (EGFR) signaling, revealed both enhanced and weakened affinity in the interactome of RSK1:PDZ domain interactions (Gógl et al, 2019). In addition, phage display analysis of the Scribble and DLG1 PDZ domains with unphosphorylated or phosphomimetic peptides of endogenous PDZ ligand sequences again suggest that phosphorylation is a powerful regulatory mechanism for altering PDZ binding and affinity of cellular targets (Sundell et al, 2018).…”