The structure of endothiapepsin complexed with a Phe-Tyr reduced-bond inhibitor at 1.35 Å resolution

Abstract

Endothiapepsin is a typical member of the aspartic proteinase family. The catalytic mechanism of this family is attributed to two conserved catalytic aspartate residues, which coordinate the hydrolysis of a peptide bond. An oligopeptide inhibitor (IC50 = 0.62 µM) based on a reduced-bond transition-state inhibitor of mucorpepsin was co-crystallized with endothiapepsin and the crystal structure of the enzyme-inhibitor complex was determined at 1.35 Å resolution. A total of 12 hydrogen bonds between the inhibitor and the active-site residues were identified. The resulting structure demonstrates a number of novel subsite interactions in the active-site cleft.

Keywords: aspartic proteases; endothiapepsin; inhibitor; transition state.

Figure 1

The suggested catalytic mechanism of endothiapepsin. A water molecule bound to Asp32 and Asp215 nucleophilically attacks the scissile-bond carbonyl. A tetrahedral intermediate is then generated and stabilized by hydrogen bonds. The C—N bond is cleaved by transferring a proton to the amino group. Hydrogen bonds are indicated by dotted lines.

Figure 2

The chemical structure of the inhibitor (FRY) co-crystallized with endothiapepsin. The sequence of the oligopeptide is Ser-Leu-Phe-His-PheTyr-Thr-Pro. There is a reduced peptide bond (indicated by an asterisk) between Phe and Tyr, i.e. the carbonyl group is replaced by a methylene –CH₂– moiety.

Figure 3

A cluster of crystals of endothiapepsin complexed with the inhibitor obtained by the hanging-drop method. The single-crystal fragment used for data collection had dimensions of approximately 200 × 100 × 50 µm.

Figure 4

2F _o − F _c map of the refined inhibitor bound in the active site contoured at 1.0 r.m.s.. Most parts of the inhibitor fitted the map quite well except for two small parts of the end residues.

Figure 5

A structural comparison of the inhibitors FRY and H256. The C atoms of the FRY inhibitor are coloured green, while those of H256 are coloured pink.

Figure 6

The enzyme and the residues which form hydrogen bonds to the inhibitor. The inhibitor conformation is stabilized by these 12 hydrogen bonds, which are indicated by purple dotted lines. The C atoms of the inhibitor are coloured green, while the C atoms of the enzyme residues are coloured yellow. The thin tube shows the tertiary structure of the enzyme.