Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design

PADs are calcium-dependent enzymes that use a nucleophilic cysteine to hydrolyze guanidinium groups on arginine residues to form citrulline (Figure 1A, Figure S1A). This reaction, termed citrullination or deimination, results in the loss of positive charge, thereby affecting protein function and altering protein–protein and protein–nucleic acid interactions.(1-4) Humans encode five PADs, designated PADs 1–4 and PAD6, which are highly homologous both within and between species (44–58% identity between human PADs). While PAD4 binds five calciums per monomer, none of these metal ions directly participate in catalysis. Nevertheless, they are critical for upregulating enzymatic activity by at least 10 000-fold.(5) Despite their high homology, the five enzymes in this family are nonredundant and regulate numerous cellular processes including pluripotency,(3, 6) myelination,(7) gene transcription,(8) kinase signaling,(9) antigen generation,(10) and neutrophil(11) and macrophage(12) extracellular trap (NET and MET) formation. Dysregulated PAD activity, most notably PAD2 and PAD4, is associated with multiple inflammatory diseases (e.g., rheumatoid arthritis) as well as cancer,(13) and PAD inhibitors, such as Cl-amidine and BB-Cl-amidine, show efficacy in multiple preclinical animal models of disease.(14-20