Structural and mechanistic analysis of two prolyl endopeptidases: role of interdomain dynamics in catalysis and specificity

Abstract

Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing insights into the mode of action of these enzymes. The structure of the Sphingomonas capsulata PEP, solved and refined to 1.8-A resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound PEP from Myxococcus xanthus was crystallized (1.5-A resolution) in a closed form. Comparative analysis of the two structures highlights a critical role for the domain interface in regulating interdomain dynamics and substrate specificity. Structure-based mutagenesis of the M. xanthus PEP confirms an important role for several interfacial residues. A salt bridge between Arg-572 and Asp-196/Glu-197 appears to act as a latch for opening or closing the didomain enzyme, and Arg-572 and Ile-575 may also help secure the incoming peptide substrate to the open form of the enzyme. Arg-618 and Asp-145 are responsible for anchoring the invariant proline residue in the active site of this postproline-cleaving enzyme. A model is proposed for the docking of a representative substrate PQPQLPYPQPQLP in the active site, where the N-terminal substrate residues interact extensively with the catalytic domain, and the C-terminal residues stretch into the propeller domain. Given the promise of the M. xanthus PEP as an oral therapeutic enzyme for treating celiac sprue, our results provide a strong foundation for further optimization of the PEP's clinically useful features.

Fig. 1.

Tertiary structures of MX and SC PEPs [drawn and rendered with pymol (Delano Scientific, San Carlos, CA)]. (A) Side view of inhibitor-bound MX PEP, represented by ribbon diagrams. The bound inhibitor Z-Ala-prolinal is colored in magenta. The inhibitor interacts only with the front half of the propeller domain. The two domains are covalently connected by two linkers consisting of residues 67–70 (marked as 1) and 407–409 (marked as 2). (B) Side view of the open SC structure. The two connecting strands between the domains are numbered as 1 (Ile-106 ∼ Glu-109, in blue) and 2 (Thr-453 ∼ Pro-449, in yellow). The C-terminal His₆-tag is fitted into an α-helix trailing on the right, in red.

Fig. 2.

MX interactions mapping. Residues (Y453, N534, S533, H651, W574, Y578, R618, D145, Y170, and T573) involved in the inhibitor-binding pocket are labeled in black, and their corresponding hydrogen-bond network is indicated by blue dashed lines. Z-Ala-prolinal is colored in orange. Additional residues (V458, G532, R572, I575, D196, and E197) and interactions of interest are indicated in the map labeled in gray and are further examined through mutagenesis. Notably, two sets of interface residues, D145 (from the propeller domain) with R618 (from the catalytic domain) and D196, E197 (propeller) with R572 (catalytic), contribute to the domain–domain interactions by forming salt bridges and hydrogen bonds.

Fig. 3.

Open structure of the SC PEP stabilized by the neighboring molecule's His₆-tag through its interaction with the catalytic domain. The α-helix at the mouth region of the catalytic domain interacts extensively with the incoming peptide (cyan).

Fig. 4.

Docking model of MX PEP. MX is docked with PQPQLPYPQPQLP. The peptide is in a green/red/blue color configuration. The active-site serine is labeled. Residues D196 and R572, which form a salt bridge at the mouth of the didomain enzyme, are also shown.