Fig. 4: The cryptic binding pocket provides selectivity of CKPs for HTRA1.
From: Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region
a Selectivity of CKPs within the HTRA family. At 2 µM concentration the parent 3B3 and affinity-matured CKPs only inhibit HTRA1PD/PDZ but not HTRA2-4PD/PDZ in the H2-OPT enzyme assay. The 7mer activity-based probe (ABP) was used as a positive control inhibitor. Values are the mean ± S.D. of at least three independent experiments. There was no statistically significant reduction in the activities of HTRA2-4PD/PDZ by the CKPs vs DMSO controls (two-sided, unpaired Student’s t test). HTRA1PD/PDZ was significantly inhibited by CKPs (P < 0.0001) except by the CPI scaffold. All HTRA proteases were significantly inhibited by the ABP (P = 0.0003 for HTRA4PD/PDZ and P < 0.0001 for HTRA1-3PD/PDZ). b SPR sensorgrams showing binding kinetics of 1C10 (1 µM) to HTRA family members (data are representative of three independent experiments). c Sequence alignment of trypsin fold serine proteases of subfamilies S1C (HTRA proteases) and of S1A, clan PA (Hepsin, plasmin etc.). HTRA proteases DegS, DegP and DegQ of Escherichia coli (strain K12), DegP9 of Arabidopsis thaliana (plant), HTRA2 of Drosophila melanogaster (fruit fly), HTRA1 of Oryzias latipes (Japanese rice fish), HTRA1 of Xenopus laevis (African clawed frog) and HTRA1 of Mus musculus (mouse). The canonical cysteine residues [C42-C58] of S1A proteases (yellow) are replaced with glycine and valine in HTRA proteases (green). Conserved residues within the two subfamilies are highlighted in gray and those conserved across both subfamilies in blue; the catalytic histidine is in purple. The gatekeeper residue V221 of HTRA1 is indicated by an arrow. Sequence alignment was carried out by using UniProt78. d The region of the cryptic pocket (dotted circle) in the 3B3:HTRA1PD(SA) complex (HTRA1 as brown cartoon) with 3B3 pocket residue Y27 in orange sticks. The gatekeeper residue V221 (magenta; in “open” position) and G204 (magenta sticks) of the β-strand descending from LoopA correspond to the canonical disulfide-forming residues [C58] and [C42], respectively (catalytic H220 is in yellow). e The canonical [C42-C58] disulfide bridge of thrombin (light blue cartoon) (PDB: 3LU9 with bound PAR-1 peptide removed) takes up the space where the cryptic pocket of HTRA1 is located (indicated by dotted circle and superimposed Y27 of 3B3).
