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

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Protein interactions are crucial for cellular functions.
• Small linear motifs binding to globular domains mediate many protein interactions.
• Quantifying motif specificity requires measuring binding affinities to all potential target domains.

Purpose of the Study:

• To develop a high-throughput method for quantifying domain-motif binding affinities.
• To assess the PDZome recognition specificity of viral motifs from human papillomavirus E6 oncoproteins.

Main Methods:

• Development of the high-throughput holdup assay, a chromatographic technique.
• Benchmarking the assay on 210 known PDZ-peptide pairs.
• Measurement of binding affinities for two viral motifs against 209 PDZ domains.

Main Results:

• The holdup assay can measure up to 1,000 domain-motif binding affinities per day.
• Detailed binding profiles were obtained for two viral motifs against 79% of the human PDZome.
• Sharply sequence-dependent binding profiles quantitatively described motif recognition specificity.

Conclusions:

• The high-throughput holdup assay is an effective tool for quantifying domain-motif binding specificities.
• This approach provides quantitative insights into viral motif recognition within the human PDZome.
• The method has broad applicability for studying domain-ligand interactions and interactome specificities.