CRYSTAL-STRUCTURE OF AN OLIGOMER OF PROTEOLYTIC ZYMOGENS - DETAILED CONFORMATIONAL-ANALYSIS OF THE BOVINE TERNARY COMPLEX AND IMPLICATIONS FOR THEIR ACTIVATION

The pancreas of ruminants secretes a 100 kDa non-covalent ternary comp lex of the zymogen of a metalloexopeptidase, carboxypeptidase A, and t he proforms of two serine endopeptidases, chymotrypsin C and proteinas e E. The crystal structure of the bovine complex has been solved and r efined to an X-factor of 0.192 using synchrotron radiation X-ray data to 2.35 Angstrom resolution. In this heterotrimeric complex, the 403 r esidue procarboxypeptidase A takes a central position, with chymotryps inogen C and proproteinase E attached to different surface sites of it . The procarboxypeptidase A subunit is composed of the active enzyme p art and the 94 residue prodomain, similar to the monomeric porcine hom ologous form. The 251 residue subunit chymotrypsinogen structure, the first solved of an anionic (acidic pi) chymotrypsinogen, exhibits char acteristics of both chymotrypsinogen A and elastases, with a potential specificity pocket of intermediate size (to accommodate apolar medium -sized residues) although not properly folded, as in bovine chymotryps inogen A; this pocket displays a ''zymogen triad'' characteristic for zymogens of the chymotrypsinogen family, consisting of three non-catal ytic residues (one serine, one histidine, and one aspartate) arranged in a fashion similar to the catalytic residues in the active enzymes. Following the traits of this family, the N terminus is clamped to the main molecular body by a disulphide bond, but the close six residue ac tivation segment is completely disordered. The third zymogen, the 253 residue proproteinase E, bears close conformational resemblance to act ive porcine pancreatic elastase; its specificity pocket is buried, dis playing the second ''zymogen triad''. Its five N-terminal residues are disordered, although the close activation site is fixed to the molecu lar surface. The structure of this native zymogen displays large confo rmational differences when compared with the recently solved crystal s tructure of bovine subunit III, an N-terminally truncated, non-activat able, proproteinase E variant lacking the first 13 residues of the nat ive proenzyme. Most of the prosegment of procarboxypeptidase A and its activation sites are buried in the centre of the oligomer, whilst the activation sites of chymotrypsinogen C and proproteinase E are surfac e-located and not involved in intra or inter-trimer contacts. This org anization confers a functional role to the oligomeric structure, estab lishing a sequential proteolytic activation for the different zymogens of the complex. The large surface and number of residues involved in the contacts among subunits, as well as the variety of non-bonded inte ractions, account for the high stability of the native ternary complex . (C) 1997 Academic Press Limited.