Quantifying domain-ligand affinities and specificities by high-throughput holdup assay.
Vincentelli R, Luck K, Poirson J, Polanowska J, Abdat J, Blémont M, Turchetto J, Iv F, Ricquier K, Straub ML, Forster A, Cassonnet P, Borg JP, Jacob Y, Masson M, Nominé Y, Reboul J, Wolff N, Charbonnier S, Travé G
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.Lire l‘article