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Nat Methods. 2015 Aug;12(8):787-93. doi: 10.1038/nmeth.3438. Epub 2015 Jun 8.

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay.

Author information

1
Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS)-Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France.
2
quipe labellisée Ligue 2015, UMR 7242, CNRS-Université de Strasbourg, École Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
3
Unité 1068 Institut national de la santé et de la recherche médicale (INSERM), UMR 7258, CNRS-Aix-Marseille Université, Centre de Recherche en Cancérologie de Marseille, Institut Paoli-Calmettes, Marseille, France.
4
Département de Biologie Structurale et Chimie, Unité de Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur, UMR 3528, CNRS, Paris, France.
5
Département de Virologie, Unité de Génétique Moléculaire des Virus à ARN, Institut Pasteur, UMR 3569, CNRS, Université Paris Diderot, Paris, France.

Abstract

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.

PMID:
26053890
PMCID:
PMC4521981
DOI:
10.1038/nmeth.3438
[Indexed for MEDLINE]
Free PMC Article

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