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

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.
Mesh Terms:
Amino Acid Motifs, Chromatography, DNA-Binding Proteins, High-Throughput Screening Assays, Humans, Kinetics, Ligands, Oncogene Proteins, Viral, PDZ Domains, Protein Conformation, Protein Interaction Mapping, Proteins, Proteome, Repressor Proteins, Systems Biology
Nat Methods
Date: Aug. 01, 2015
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