Covalent Modification of Bromodomain Proteins by Peptides Containing a DNA Damage‐Induced, Histone Post‐Translational Modification

An electrophilic 5-methylene-2-pyrrolone modification (K-MP) is produced at lysine residues of histone proteins in nucleosome core particles upon reaction with a commonly formed DNA lesion (C4-AP). The nonenzymatic K-MP modification is also generated in the histones of HeLa cells treated with the antitumor agent, bleomycin that oxidizes DNA and forms C4-AP. This nonenzymatic covalent histone modification has the same charge as the N-acetyllysine (K-Ac) modification but is more electrophilic. In this study we show that K-MP-containing histone peptides are recognized by, and covalently modify bromodomain proteins that are K-Ac readers. Distinct selectivity preferences for covalent bromodomain modification are observed following incubation with K-MP-containing peptides of different sequence. MS/MS analysis of 3 covalently modified bromodomain proteins confirmed that Cys adduction was selective. The modified Cys was not always proximal to the K-Ac binding site, indicating that K-MP-containing peptide interaction with bromodomain protein is distinct from the former. Analysis of protein adduction yields as a function of bromodomain pH at which the protein charge is zero (pI) or cysteine solvent accessible surface area are also consistent with non-promiscuous interaction between the proteins and electrophilic peptides. These data suggest that intracellular formation of K-MP could affect cellular function and viability by modifying proteins that regulate genetic expression.