Fig. 3: Divalent metal ions modulate the proteolytic activity of HtrA2.

a Proteolytic assay using β-casein shows that Mg²⁺ and Ca²⁺ enhanced the proteolytic activity of HtrA2, while supplementation of Zn²⁺ and Cu²⁺ strongly inhibited proteolytic activity. Cleavage assays were done as biological triplicates, yielding similar results (Supplementary Fig. 9). b Comparison of the proteolytic efficiency of HtrA2 towards β-casein without or with divalent metal ions. Cleavage is expressed as percentage of β-casein cleaved after 40 minutes of incubation with HtrA2. Bar colors correspond to the color assigned to each metal ion in (a). c Scheme of the fluorescence assay using an activating peptide and a reporter peptide. d Fluorescence assay showing activity of HtrA2 with an HtrA2 activating peptide (DD-PDZOpt, 50 µM) using varying reporter peptide concentrations as indicated. Data is representative of three replicate experiments. e Analysis of the catalytic efficiency (k_cat/K_M) of the assay shown in (d) in the absence or presence of divalent metal ions as indicated. f Determination of the apparent K_D (K_D,app) of the activator peptide using varying peptide concentrations. n.d. indicates not determined. Values are the averages of n  =  3 individual repeats, and error bars indicate the SD of these replicates. g Fluorescence assay showing activity of HtrA2 using varying HtrA2 activating peptide concentrations as indicated with a fixed reporter peptide concentration (20 µM). Data are representative of three replicate experiments. h Analysis of the catalytic efficiency (k_cat/K_M) of the assay shown in (g) with or without divalent metal ions. Source data are provided as a Source Data File.