Exploring the Ligand Preferences of the PHD1 Domain of Histone Demethylase KDM5A Reveals Tolerance for Modifications of the Q5 Residue of Histone 3

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.

Recognition of Histone H3 Methylation States by the PHD1 Domain of Histone Demethylase KDM5A

“…Collectively, our data suggest that H3 residues A1, R2, and K4 are the major determinants of H3 peptide binding, with smaller contributions from H3T3. Our recent study has also shown that H3Q5 is dispensable for binding as H3 Q5A peptide binds with similar affinity to WT H3 . These data suggest a finely tuned binding site with varying, methylation-dependent contributions to H3 binding and H3K4 methyl discrimination.…”

“…Our recent study has also shown that H3Q5 is dispensable for binding as H3 Q5A peptide binds with similar affinity to WT H3. 70 These data suggest a finely tuned binding site with varying, methylationdependent contributions to H3 binding and H3K4 methyl discrimination. However, the contributions of these residues to binding may not be conserved between KDM5 family PHD1 domains.…”

Chemical Biology Approaches to Identify and Profile Interactors of Chromatin Modifications

“…For example, they found binding of BRD4 to di- and triacetylated H4 peptides was detected with their optimized protocol but not using a more conventional procedure. A recent innovation coupled a peptide array to a Matrix-Assisted Laser Desorption/Ionization (MALDI) MS readout . Peptides were immobilized on a gold surface using alkanethiolate chemistry such that each spot contained one histone peptide and a reporter peptide that can be deacetylated by KDAC8.…”

“…A recent innovation coupled a peptide array to a Matrix-Assisted Laser Desorption/Ionization (MALDI) MS readout. 82 Peptides were immobilized on a gold surface using alkanethiolate chemistry such that each spot contained one histone peptide and a reporter peptide that can be deacetylated by KDAC8. A fusion protein of KDAC8 and a POI was exposed to the peptide array.…”