CRYSTALLOGRAPHIC STUDY OF A CLEAVED, NON-ACTIVATABLE FORM OF PORCINE ZYMOGEN-E

The crystal structure of a cleaved form of porcine zymogen E has been solved by molecular replacement using the bovine procarboxypeptidase A -S6 subunit III structure as search model. Crystallographic refinement using simulated annealing and energy minimization techniques resulted in a final R-factor of 0.189 for all data between 8 and 2.3 Angstrom resolution. The zymogen E three-dimensional model is very close to tha t of bovine subunit III and represents the second member of the zymoge n E family for which the crystal structure is known. The two structure s indicate that, in contrast to trypsinogen and chymotrypsinogen, zymo gens of this family are highly organized molecules. The amino acid seq uence of zymogen. E has only been determined for the first 40 residues . Based on the electron density map, we have introduced six sequence c hanges relative to subunit III. Out of the 11 residues in the activati on peptide, only the first six present well matching electron density; they are connected to the rest of the zymogen by an unexpected Cys1-C ys122 disulphide bridge (according to the bovine chymotrypsinogen A nu mbering system). Amino acid sequencing of protein solutions both from dissolved crystals and from the initial stock clearly indicated that t he Val17-Asn18 bond had been cleaved during the crystallization proces s. This result adds weight to the assumption that the autolysis of the bovine zymogen E gives rise to subunit III and that this maybe a regu latory mechanism for protease E activity. (C) 1995 Academic Press Limi ted