Fig 2: PAD-PF2 potently inhibits PAD1-4 isoforms in a Ca2+-dependent manner.a PAD-PF2 (open circles) inhibits PAD4-mediated citrullination of peptide substrate Atto655-(Ser-Arg-Gly-Ala)3 employing a fluorescence quenching (FQ) assay in the presence of 0.4 mM Ca2+. Data points and error bars represent the mean and SD of three independent experiments in duplicate. IC50 = 42.7 nM, pIC50 = 7.37 (0.03), mean (SD), n = 3 independent experiments. The enantiomer of PAD-PF2 (ent-PAD-PF2, open squares) was less potent. IC50 = 26.0 µM, pIC50 = 4.59 (0.45), mean (SD), n = 3 independent experiments. b PAD-PF2 induces a thermal stabilization of PAD2 (open circles, EC50 = 2.04 µM) and PAD4 (open squares, EC50 = 1.95 µM) protein. Individual data points shown for a duplicate determination. c–f Inhibition of the respective PAD isoform by PAD-PF2 determined using the GDH-enzyme coupled assay in the presence of 0.25 mM Ca2+. Individual data points shown for a duplicate determination along with the fitted IC50 curves (line). c PAD1, IC50 = 109 nM. d PAD2, IC50 = 28.5 nM. e PAD3, IC50 = 106 nM. f PAD4, IC50 = 24.0 nM. g, h Calcium dependence of PAD2 or PAD4 inhibition by PAD-PF2. Inhibition was determined using the GDH enzyme-coupled assay. Data points from duplicate experiments performed at high (open squares) and low (open circles) Ca2+ concentrations are plotted together, and the fitted IC50 curves are shown (line). g The determined IC50 values for PAD2 inhibition were 27.9 nM and 3.09 µM (0.25 and 1.5 mM Ca2+, respectively). h The determined IC50 values for PAD4 inhibition were 20.1 nM and 694 nM (0.25 and 1.5 mM Ca2+, respectively). Source data are provided as a Source Data file.

Fig 3: PAD substrate specificity at the P4–P2 positions toward natural amino acids.(A) Substrate specificity profiles of PAD1, PAD2, PAD3, and PAD4 were evaluated at P2, P3, and P4 positions using the Ac-P4-P3-P2-Arg-ACC HyCoSuL library, where the P1 position was fixed as arginine. The relative enzymatic preferences for natural amino acids at each position were visualized as color-coded heat maps, with the most intense color representing 100% relative activity, normalized to the most efficiently cleaved substrate for each PAD isoform. (B) PAD4 substrate specificity at P4–P2 was further assessed under two distinct conditions: high CaCl2 concentration (maximally activating) and low CaCl2 concentration supplemented with heparin (allosteric modulator). The results are displayed as grayscale heat maps, where black indicates the highest relative activity for a given amino acid at each position. Arginine at the P2 position was excluded from the analysis because the Ac-P4-P3-Arg-Arg-ACC substrate underwent rapid citrullination at P2. The resulting product (Ac-P4-P3-Cit-Arg-ACC) was more efficiently recognized by trypsin than the original substrate was. Additionally, proline at the P3 position was excluded because trypsin does not tolerate this residue at that site.