High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

Abstract

The conformation of the synthetic renin inhibitor CP‐69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X‐ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP‐69,799 is an oligopeptide transition‐‐state analogue inhibitor that contains a new dipeptide isostere at the P1‐P1′ position. This dipeptide isostere is a nitrogen analogue of the well‐explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1′ C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3′ pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3′. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.

Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design.

“…No subdomain displacements were observed in cathepsin D upon inhibitor binding, unlike the case of pepsin (27) and endothiapepsin (28).…”

“…The crystal structure of porcine pepsin was used as the search molecule (22). The final R factor was 18.8% for data from 10.0 to 2.5 A resolution for 28 (Fig. 1): an N-terminal domain (residues 1-188), a C-terminal domain (residues 189-346), and an interdomain, anti-parallel /3sheet composed of the N terminus (residues 1-7), the C terminus (residues 330-346), and the interdomain-linking residues (160-200).…”

Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: Implications for the design of antifungal agents

“…The approximate relative orientation of the segment of residues between 190 and 301 (pepsin numbering) is an important element in the structural variability observed within the carboxy domain of the monomeric aspartic proteinases (Sali et al, 1989(Sali et al, , 1992Abad-Zapatero et al, 1990;Sielecki et al, 1990). After the rigid body domain 1 (RBI, 1-189 and 301-326) has been superimposed between a certain pair, the relative orientation of rigid body domain 2 can range widely among the different enzymes.…”

Could domain movements be involved in the mechanism of trypsin‐like serine proteases?

“…Many enzymes possess distinct domains in their superstructure and in several instances, relative movement of the domains is thought to be important for the entrance and exit of substrates and products [l], or the stressing of bonds that are required to be broken [2]. The trypsin-like serine proteases, a large family of peptide and protein cleaving enzymes, are superficially promising candidates for one to suspect such a contribution to their mechanism.…”