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Proteome‐wide analysis of phospho‐regulated PDZ domain interactions

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 proteo...

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Autores principales: Sundell, Gustav N, Arnold, Roland, Ali, Muhammad, Naksukpaiboon, Piangfan, Orts, Julien, Güntert, Peter, Chi, Celestine N, Ivarsson, Ylva
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100724/
https://www.ncbi.nlm.nih.gov/pubmed/30126976
http://dx.doi.org/10.15252/msb.20178129
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author Sundell, Gustav N
Arnold, Roland
Ali, Muhammad
Naksukpaiboon, Piangfan
Orts, Julien
Güntert, Peter
Chi, Celestine N
Ivarsson, Ylva
author_facet Sundell, Gustav N
Arnold, Roland
Ali, Muhammad
Naksukpaiboon, Piangfan
Orts, Julien
Güntert, Peter
Chi, Celestine N
Ivarsson, Ylva
author_sort Sundell, Gustav N
collection PubMed
description 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.
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spelling pubmed-61007242018-08-27 Proteome‐wide analysis of phospho‐regulated PDZ domain interactions Sundell, Gustav N Arnold, Roland Ali, Muhammad Naksukpaiboon, Piangfan Orts, Julien Güntert, Peter Chi, Celestine N Ivarsson, Ylva Mol Syst Biol Methods 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. John Wiley and Sons Inc. 2018-08-20 /pmc/articles/PMC6100724/ /pubmed/30126976 http://dx.doi.org/10.15252/msb.20178129 Text en © 2018 The Authors. Published under the terms of the CC BY 4.0 license This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods
Sundell, Gustav N
Arnold, Roland
Ali, Muhammad
Naksukpaiboon, Piangfan
Orts, Julien
Güntert, Peter
Chi, Celestine N
Ivarsson, Ylva
Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
title Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
title_full Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
title_fullStr Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
title_full_unstemmed Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
title_short Proteome‐wide analysis of phospho‐regulated PDZ domain interactions
title_sort proteome‐wide analysis of phospho‐regulated pdz domain interactions
topic Methods
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100724/
https://www.ncbi.nlm.nih.gov/pubmed/30126976
http://dx.doi.org/10.15252/msb.20178129
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