Abstract
Understanding the ligand preferences of epigenetic reader domains enables identification of modification states of chromatin with which these domains associate and can yield insight into recruitment and catalysis of chromatin-acting complexes. However, thorough exploration of the ligand preferences of reader domains is hindered by the limitations of traditional protein-ligand binding assays. Here, we evaluate the binding preferences of the PHD1 domain of histone demethylase KDM5A using the protein interaction by SAMDI (PI-SAMDI) assay, which measures protein-ligand binding in a high-throughput and sensitive manner via binding-induced enhancement in the activity of a reporter enzyme, in combination with fluorescence polarization. The PI-SAMDI assay was validated by confirming its ability to accurately profile the relative binding affinity of a set of well-characterized histone 3 (H3) ligands of PHD1. The assay was then used to assess the affinity of PHD1 for 361 H3 mutant ligands, a select number of which were further characterized by fluorescence polarization. Together, these experiments revealed PHD1's tolerance for H3Q5 mutations, including an unexpected tolerance for aromatic residues in this position. Motivated by this finding, we further demonstrate a high-affinity interaction between PHD1 and recently identified Q5-serotonylated H3. This work yields interesting insights into permissible PHD1-H3 interactions and demonstrates the value of interfacing PI-SAMDI and fluorescence polarization in investigations of protein-ligand binding.