Citrullination in proteins of the polycomb repressor complex 1

The Polycomb repressive complexes can be considered a paradigm of chromatin-based gene regulation in animals. Two of the proteins involved in such complexes are the Ring finger protein 1 (RING1) and the intrinsically disordered protein RING1A and YY1 binding protein (RYBP), which binds to RING1 in some of the Polycomb complexes. Peptidyl arginine iminohydrolases (PADIs) are enzymes involved in the post-translational modification of arginine to citrulline. PADI4, one of the five isoforms of PADI in humans, citrullinates RING1B and RYBP at specific arginines at their C-terminal (Arg231) and N-terminal (Arg53) regions, respectively. In this work, we studied in vitro and in silico whether isolated peptides from each protein, encompassing those wild-type arginines and their citrulline-modified counterparts, were capable of binding to PADI4. The isolated peptides, either in wild-type or modified form, were monomeric and disordered as indicated by far-UV CD and NMR. The affinity constants of peptides for PADI4, as measured by fluorescence, biolayer interferometry (BLI) and isothermal titration calorimetry (ITC), were in the low micromolar range (dissociation constants around 10-20 μM), and the affinities were smaller for the citrullinated variants. Molecular docking simulations showed that the isolated peptides from each protein, either in wild-type form or modified variants, targeted the active site of PADI4, corresponding to the catalytic residue Cys645. These findings suggest that the isolated peptides were capable of binding to the enzyme with affinities similar to those of intact RING1B or RYBP, and that citrullination of the arginines diminished the affinity for the enzyme.