Fig. 5: Deletion or perturbation of LoopA diminishes catalytic activity within the HTRA family.

a Enzymatic activity of wildtype HTRA1^PD, HTRA2^PD/PDZ, and HTRA3^PD/PDZ compared to their ΔLoopA mutants; Fab15H6.v4 and the control Fab33 did not modify the mutant activities. b Size-exclusion chromatography profiles of HTRA1^PD (blue), HTRA1^PD-∆LoopA (purple), HTRA2^PD/PDZ (light purple) and HTRA2^PD/PDZ-∆LoopA (brown) indicate formation of trimers (protein standards in grey). c Enzymatic activity of LoopA chimeras of HTRA1^PD in the presence or absence of Fab15H6.v4. d In-vitro DKK3 cleavage assay using purified HTRA1^PD or HTRA1^PD-ΔLoop mutant in the presence or absence of Fab15H6.v4. e In-vitro DKK3 cleavage assay using wildtype HTRA1^PD and the LoopA swap chimera HTRA1^PD-LoopA^HTRA2. f Design and enzymatic activity of single amino acid substitutions within LoopA of HTRA1^PD and of GSG/GSGSG linkers. g Control gels showing individual proteins incubated under identical conditions as used for cleavage assays in d, e Asterisk (*) in (d, e, g) indicates contaminant in the Fab33 preparations that overlaps with the cleaved DKK3 band. Bar graphs in a, c, f are presented as the mean ± S.D. of three independent experiments. Images in d, e, g as well as chromatogram in b are representative of two independent experiments. For experiments in a, b the HTRA2 and HTRA3 constructs comprised the protease and PDZ domains (HTRA2/3^PD/PDZ). Source data are provided as a Source Data file.