Journal
NATURE METHODS
Volume 12, Issue 8, Pages 787-U154Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.3438
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Funding
- CNRS
- Universite de Strasbourg, Infrastructures Biologie Sante et Agronomie (Ibisa)
- Association pour la Recherche contre le Cancer (ARC) [3171]
- Agence Nationale de la Recherche [ANR-MIME-2007 EPI-HPV-3D]
- US National Institutes of Health (NIH) [R01CA134737]
- Ligue contre le Cancer and Alsace contre le Cancer
- Region Alsace
- ARC
- Ligue Regionale contre le Cancer
- La Ligue Contre le Cancer
- Institut paoli-Calmettes
- Canceropole PACA
- SIRIC [6038]
- NATIONAL CANCER INSTITUTE [R01CA134737] Funding Source: NIH RePORTER
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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.
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