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 A resolution. The zymogen E three-dimensional model is very close to that of bovine subunit III and represents the second member of the zymogen E family for which the crystal structure is known. The two structures indicate that, in contrast to trypsinogen and chymotrypsinogen, zymogens of this family are highly organized molecules. The amino acid sequence of zymogen E has only been determined for the first 40 residues. Based on the electron density map, we have introduced six sequence changes relative to subunit III. Out of the 11 residues in the activation peptide, only the first six present well matching electron density; they are connected to the rest of the zymogen by an unexpected Cys1-Cys122 disulphide bridge (according to the bovine chymotrypsinogen A numbering system). Amino acid sequencing of protein solutions both from dissolved crystals and from the initial stock clearly indicated that the Val17-Asn18 bond had been cleaved during the crystallization process. 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 regulatory mechanism for protease E activity.
Pignol
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Laboratoire de Cristallographie et de Cristallogénèse des Protéines Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), Grenoble, France.
Granon
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Chapus
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Fontecilla-Camps
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JC
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D004792:Enzyme Precursors / Q000648:ultrastructure*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000648:ultrastructure*; D013552:Swine
D004792:Enzyme Precursors; D012697:Serine Endopeptidases; C028736:zymogen E