Fig. 3: Crystal structure of the 3B3:HTRA1PD(SA) complex shows the cryptic pocket and non-competent active site.
From: Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region
a Overview of 3B3:HTRA1PD(SA) complex (HTRA1PD(SA), protease domain with active site S328A mutation). Surface representation of the HTRA1 protomers (brown, green and blue) with the three 3B3 peptides (red, cartoon) bound to the S’ region between the catalytic triad (D250, H220, S328A in yellow) and LoopA. The approximate path of the substrate binding cleft and the S and S′ subsite regions are indicated. b Close-up of the binding interface between 3B3 (chain I) with HTRA1 protomer (chain A, brown) and minor contacts with HTRA1-chain C. Contacts within 4 Å of 3B3 on HTRA1 (surface representation) are shown in cyan and are labeled in black italics (catalytic H220 and S328A in yellow). The contact residues on 3B3 (within 4 Å from HTRA1) are labelled in red and the two main binding loops are in salmon (Loop5) and green (Loop2). 3B3 also makes a minor contact to the neighboring HTRA1 protomer (chain C). c and d The open Y27-pocket of the 3B3:HTRA1PD(SA) complex (3B3 contact residues in cyan as in (b), gatekeeper V221 in magenta, catalytic H220 in yellow) as surface representation (c) or with key pocket residues as sticks (d); the 3B3 residues Y27 and W30 are shown as salmon sticks. e and f The closed Y27-pocket of apo-HTRA1 (PDB: 3TJN-chain B38) with HTRA1 in surface representation (e) or with key pocket residues as sticks (f) (HTRA1 in green). The superimposed 3B3 residues Y27 and W30 (as salmon sticks) clash with the closed pocket (e), which is filled by V221 (magenta). In the closed-to-open transition (f and d) the gatekeeper residue V221 (magenta) moves away to open the pocket and allowing Y27 insertion. In the open pocket the gatekeeper V221 is in almost the same location as V222 in the closed conformation (dotted circles). Movements of the residues, including those of the β-strand (A202-S205), are indicated by arrows. g Interactions of 3B3 residues (sticks) that stabilize the non-competent active site conformation of HTRA1. The 3B3 residues Y26 and P34 contact HTRA1-Y325 (cyan) which stabilizes a distorted conformation of the oxyanion hole (dotted circle) and 3B3 residues A28 and T29 stabilize the flipped conformation of L345 (cyan), which occludes the S1 specificity pocket. The HTRA1-Loop2 residues T348 and A349 are not resolved in the structure and are shown as dotted line. h Comparison of the non-competent active site conformations of the 3B3:HTRA1 complex (red) and the Fab15H6.v4:HTRA1 complex (PDB: 7SJN-chain A; yellow) with the competent conformation of apo-HTRA1 (PDB: 3TJN-chain B; green). The H220 and L345 residues are shown as sticks and the S1 pocket and oxyanion hole are indicated as dotted lines.
