Fig. 4: Fab15H6.v4 binds to HTRA1 LoopA epitope with high specificity.

a SPR binding kinetics show no interaction between Fab15H6.v4 and HTRA1-∆LoopA mutant. Overview of the co-crystal structure of Fab15H6.v4 with LoopA peptide (center). b Detail of co-crystal structure showing an excellent alignment of the LoopA peptide (orange) with the LoopA of the cryo-EM structural model (blue). In the co-crystal structure the LoopA is exclusively contacted by the Fab heavy chain (CDR1-3, red). c Side chain comparison between LoopA peptide of the co-crystal structure (orange) and LoopA of the cryo-EM structural model (blue) with key residues labeled in red. d Key residues between LoopA and Fab15H6.v4 based on the co-crystal structure include R190, L192, P193 and R197 (labeled red). e SPR binding kinetics of immobilized Fab15H6.v4 to LoopA peptides derived from HTRA family members. f, g SPR binding kinetics experiments show no interaction between Fab15H6.v4 and the chimeric proteins HTRA1-LoopA^HTRA2, HTRA1-LoopA^HTRA3 and HTRA1-LoopA^HTRA4. h Alignment of partial protease domain sequences of HTRA family members highlighting the poor conservation of LoopA. Red stars indicate residues important for Fab interaction (see Supplementary Tables 4 and 5). Phylogram on the left based on sequence conservation of the HTRA1-4 protease domains. Boundaries of the protease domains are indicated on the left. SPR kinetic data in (a, e–g) are presented as the mean ± S.D. of three independent experiments.