Fig. 3: Meprin α oligomers are proteolytically and thermally stable compared to lower-stoichiometry variants.

a Periodic arrangement of the active site and pro-domain are highlighted as yellow in the surface representation of an idealised meprin α helical segment. b Example curve representing a series of first-order rates (determined by fitting the linear region of fluorescence versus time) at a given meprin concentration (each point is the mean of n = 2 technical repeats; each curve was measured in n = 3 independent experiments). The k_cat/K_m is determined from this graph for several meprin concentrations and in triplicate c First order rate constants (k_cat/K_m) of meprin α and variants for small fluorogenic peptide cleavage. Oligomeric state does not appear to affect rate of cleavage of small molecule substrate (n = 3 independent experiments with n = 2 technical repeats). Statistical test of one-way ANOVA, n.s. is not significant, Tukey’s test for multiple comparisons. d Apparent potency measured as the inhibitory constant (IC₅₀) of meprin α inhibitors. Determined by fitting and normalising the linear region of fluorescence versus time, in the presence of varying amount of inhibitor at a fixed meprin α concentration (each point is the mean of n = 2 to 4 technical repeats across n = 3 independent experiments). e Globular proteinaceous inhibitor, murine fetuin-B, and small molecule compound 10d were unaffected by oligomeric state (determined by fitting 3d, i.e., n = 3 independent experiments). Statistical test of one-way ANOVA, n.s. is not significant, Tukey’s test for multiple comparisons. f Proteolytic stability of meprin α and variants against trypsin and plasmin. Meprin α oligomers were more stable compared to lower-stoichiometric variants. g Meprin α thermal stability measured by nanoDSF (n = 3 independent experiments). Meprin α possess superior thermal stability compared to variants that lack either the disulphide bridge (most drastic) or helical interface interactions. One-way ANOVA reports statistically significant difference for each comparison (i.e., all versus all; p < 0.0001, ****). For all panels, each point is the mean and error bars represent the standard deviation (±σ). Source data are provided as a Source Data file.