Fig. 6: Transfer of the Fab15H6.v4 allosteric inhibition mechanism to HTRA2 and HTRA3.

a Enzymatic activity of wildtype HTRA2^PD/PDZ (pink) and HTRA3^PD/PDZ (blue) compared to the chimeric HTRA2^PD/PDZ-LoopA^HTRA1 (green) and HTRA3^PD/PDZ-LoopA^HTRA1 (purple) proteins in the presence of Fab15H6.v4 or control Fab33. b SPR binding kinetics of Fab15H6.v4 interaction with wildtype HTRA2^PD/PDZ and HTRA3^PD/PDZ and their LoopA chimeras. c, d Labeling of HTRA2^PD/PDZ and HTRA3^PD/PDZ wildtype proteins compared to the LoopA chimeras using fluorescent activity-based probe (TAMRA-ABP) in the absence or presence of Fab15H6.v4 or control Fab33. e In-vitro cleavage of DKK3 substrate using wildtype HTRA2^PD/PDZ or the chimeric HTRA2^PD/PDZ-LoopA^HTRA1 in the presence of Fab15H6.v4. No cleavage was detected using HTRA3^PD/PDZ or HTRA3^PD/PDZ-LoopA^HTRA1 (not shown) f Control gel showing individual proteins incubated under identical conditions as used for cleavage assay in e Asterisks (*) in (e, f) indicate contaminant in the Fab33 preparation that overlaps with the cleaved DKK3 band. Bar graphs in a and kinetic data in b are presented as the mean ± S.D. of three independent experiments. Images in c–f are representative of two independent experiments. For all experiments in Fig. 6 the HTRA2 and HTRA3 constructs comprised the protease and PDZ domains (HTRA2/3^PD/PDZ). Source data are provided as a Source Data file.