Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

Abstract

Crystals of the molecular complex between the esterified tripeptide fragment of pepstatin and the aspartyl proteinase penicillopepsin are isomorphous with crystals of native penicillopepsin [from Penicillium janthinellum]. The difference electron-density map at 1.8-.ANG. resolution, computed by using the amplitude differences and refined phases of reflections from the crystal of native penicillopepsin, showed the binding mode of isovaleryl-Val-Val-StaOEt, where StaOEt is the ethyl ester of statine [(4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid]. A major conformational change in penicillopepsin involving the large .beta. loop of residues from Trp-71 to Gly-83 (the so-called flap region) occurs as a result of this inhibitor binding. This structural movement provides the 1st confirmation of the importance of enzyme flexibility in the aspartyl proteinase mechanism. The 3-hydroxyl group of the Statine residue and the carbonyl O atom of the ethyl ester are situated on either side of the approximate plane containing the H-bonded carboxyl groups of Asp-33 and Asp-213. The observed binding mode of the pepstatin tripeptide fragment is similar to that predicted for the binding of good substrates with penicillopepsin.