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

Renaud Vincentelli (1) , Katja Luck (2) , Juline Poirson (2) , Jolanta Polanowska (3) , Julie Abdat (1) , Marilyne Blémont (1) , Jeremy Turchetto (1) , François Iv (1) , Kevin Ricquier (4) , Marie-Laure Straub (2, 5) , Anne Forster (2, 5) , Patricia Cassonnet (6) , Jean-Paul Borg (3) , Yves Jacob (6) , Murielle Masson (2, 5) , Yves Nominé (2, 5) , Jérome Reboul (3) , Nicolas Wolff (4) , Sebastian Charbonnier (2, 5) , Gilles Travé (2, 5)

1 AFMB - Architecture et fonction des macromolécules biologiques
2 BSC - Biotechnologie et signalisation cellulaire
3 CRCM - Centre de Recherche en Cancérologie de Marseille
4 Résonance Magnétique Nucléaire des Biomolécules
5 Equipe labellisée Ligue contre le Cancer
6 GMV-ARN (UMR_3569 / U-Pasteur_2) - Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses

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.

Domaines: Sciences du Vivant [q-bio]; Biochimie, Biologie Moléculaire; Biochimie [q-bio.BM]

Renaud Vincentelli, Katja Luck, Juline Poirson, Jolanta Polanowska, Julie Abdat, et al.. Quantifying domain-ligand affinities and specificities by high-throughput holdup assay. Nature Methods, 2015, 12 (8), pp.787-793. ⟨10.1038/nmeth.3438⟩. ⟨pasteur-02883959⟩