Open AccessQuantifying domain-ligand affinities and specificities by high-throughput holdup assay
Author(s) -
Renaud Vincentelli,
Katja Luck,
Juline Poirson,
Jolanta Polanowska,
Julie Abdat,
Marilyne Blémont,
Jeremy Turchetto,
} Francois,
Kevin Ricquier,
Marie-Laure Straub,
Anne Förster,
Patricia Cassonnet,
Jean-Paul Borg,
Yves Jacob,
Murielle Masson,
Yves Nominé,
Jérôme Reboul,
Nicolas Wolff,
Sébastian Charbonnier,
Gilles Travé
Publication year - 2015
Publication title -
nature methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 19.469
H-Index - 318
eISSN - 1548-7105
pISSN - 1548-7091
DOI - 10.1038/nmeth.3438
Subject(s) - pdz domain , affinities , binding affinities , computational biology , binding selectivity , biology , plasma protein binding , binding site , chemistry , biophysics , genetics , microbiology and biotechnology , biochemistry , receptor
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.