This study identifies functional antibodies that activate or inhibit protein arginine deiminase 4 (PAD4), a calcium-dependent enzyme involved in citrullination of proteins and the pathogenesis of rheumatoid arthritis (RA). Using unbiased antibody selection and cryogenic-electron microscopy (cryo-EM), the researchers characterized the structures of these antibodies in complex with PAD4 and revealed their mechanisms of action. The antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket, leading to changes in the active site conformation or enzyme oligomeric state. These findings highlight the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and for future therapeutic development. PAD4 is a calcium-dependent enzyme that catalyzes the hydrolysis of peptidyl arginine sidechains to citrulline in proteins. In neutrophils, PAD4-catalyzed chromatin decondensation plays a role in inducing NETosis, a form of neutrophil cell death. PAD4 and other intracellular contents are released to the extracellular space, where PAD4 can create extracellular citrullinated neoepitopes that trigger inflammatory diseases such as RA. Small-molecule PAD4 inhibitors have been developed and found to be effective in alleviating RA phenotypes in mouse models, indicating the relevance of PAD4 in RA pathology. However, these inhibitors often lack specificity and potency due to direct targeting of the enzyme's active site. Antibodies are powerful tools for capturing the dynamic nature of proteins by binding to both enzyme active sites and allosteric sites. The study used an unbiased antibody selection strategy, coupled with functional screening and cryo-EM structural analysis, to identify new conformations and mechanisms for inhibiting or activating both murine and human PAD4 in the presence of high calcium. The researchers discovered that PAD4 activity can be enhanced through antibody binding to an interface loop that promotes PAD4 dimerization while reducing disorder in the substrate-binding loop. They also discovered an inhibitory antibody that binds and restructures a helix in the calcium-binding pocket that mediates a conformational change in the active site, preventing calcium ion and substrate binding. The study identified several antibodies that modulate PAD4 dimerization, which influences enzyme activity. The results show that dimerization is important for PAD4 activity, and that the binding and activation efficacy of antibodies is influenced by dimerization of PAD4. The study also identified an inhibitory antibody that restructures the calcium-binding pocket, leading to disruption of substrate binding and loss of enzymatic activity. The researchers optimized the inhibitory antibody to improve its solubility and inhibition activity, resulting in highly specific and functional PAD4 binders that may be used to investigate PAD4 activity in mouse arthritis models and human samples. These findings provide new insights into the regulation of PAD4 and its potential as a therapeutic target for RAThis study identifies functional antibodies that activate or inhibit protein arginine deiminase 4 (PAD4), a calcium-dependent enzyme involved in citrullination of proteins and the pathogenesis of rheumatoid arthritis (RA). Using unbiased antibody selection and cryogenic-electron microscopy (cryo-EM), the researchers characterized the structures of these antibodies in complex with PAD4 and revealed their mechanisms of action. The antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket, leading to changes in the active site conformation or enzyme oligomeric state. These findings highlight the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and for future therapeutic development. PAD4 is a calcium-dependent enzyme that catalyzes the hydrolysis of peptidyl arginine sidechains to citrulline in proteins. In neutrophils, PAD4-catalyzed chromatin decondensation plays a role in inducing NETosis, a form of neutrophil cell death. PAD4 and other intracellular contents are released to the extracellular space, where PAD4 can create extracellular citrullinated neoepitopes that trigger inflammatory diseases such as RA. Small-molecule PAD4 inhibitors have been developed and found to be effective in alleviating RA phenotypes in mouse models, indicating the relevance of PAD4 in RA pathology. However, these inhibitors often lack specificity and potency due to direct targeting of the enzyme's active site. Antibodies are powerful tools for capturing the dynamic nature of proteins by binding to both enzyme active sites and allosteric sites. The study used an unbiased antibody selection strategy, coupled with functional screening and cryo-EM structural analysis, to identify new conformations and mechanisms for inhibiting or activating both murine and human PAD4 in the presence of high calcium. The researchers discovered that PAD4 activity can be enhanced through antibody binding to an interface loop that promotes PAD4 dimerization while reducing disorder in the substrate-binding loop. They also discovered an inhibitory antibody that binds and restructures a helix in the calcium-binding pocket that mediates a conformational change in the active site, preventing calcium ion and substrate binding. The study identified several antibodies that modulate PAD4 dimerization, which influences enzyme activity. The results show that dimerization is important for PAD4 activity, and that the binding and activation efficacy of antibodies is influenced by dimerization of PAD4. The study also identified an inhibitory antibody that restructures the calcium-binding pocket, leading to disruption of substrate binding and loss of enzymatic activity. The researchers optimized the inhibitory antibody to improve its solubility and inhibition activity, resulting in highly specific and functional PAD4 binders that may be used to investigate PAD4 activity in mouse arthritis models and human samples. These findings provide new insights into the regulation of PAD4 and its potential as a therapeutic target for RA