A cyclic peptide toolkit reveals mechanistic principles of peptidylarginine deiminase IV regulation

Peptidylarginine deiminase IV (PADI4, PAD4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to a lack of appropriate systems and tools. Here, we develop and apply a set of potent and selective PADI4 modulators. Using the mRNA-display-based RaPID system, we screen >10 12 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_ 3 , a cell-active inhibitor specific for the active conformation of PADI4; PADI4_ 7 , an inert binder, which we functionalise for the isolation and study of cellular PADI4; and PADI4_ 11 , a cell-active PADI4 activator. Structural studies with PADI4_ 11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work contributes to our understanding of PADI4 regulation and provides a toolkit for the study and modulation of PADI4 across (patho)physiological contexts. Walport & coworkers report cell-active, potent and selective cyclic peptide modulators for the enzyme peptidylarginine deiminase PADI4/PAD4, including an inhibitor, an activator and an affinity molecule, revealing insights into PADI4 regulation.