title: Crystal structure of an oligomer of proteolytic zymogens: detailed conformational analysis of the bovine ternary complex and implications for their activation.
abstract: The pancreas of ruminants secretes a 100 kDa non-covalent ternary complex of the zymogen of a metalloexopeptidase, carboxypeptidase A, and the proforms of two serine endopeptidases, chymotrypsin C and proteinase E. The crystal structure of the bovine complex has been solved and refined to an R-factor of 0.192 using synchrotron radiation X-ray data to 2.35 A resolution. In this heterotrimeric complex, the 403 residue procarboxypeptidase A takes a central position, with chymotrypsinogen C and proproteinase E attached to different surface sites of it. The procarboxypeptidase A subunit is composed of the active enzyme part and the 94 residue prodomain, similar to the monomeric porcine homologous form. The 251 residue subunit chymotrypsinogen structure, the first solved of an anionic (acidic pI) chymotrypsinogen, exhibits characteristics 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 chymotrypsinogen A; this pocket displays a "zymogen triad" characteristic for zymogens of the chymotrypsinogen family, consisting of three non-catalytic 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 activation segment is completely disordered. The third zymogen, the 253 residue proproteinase E, bears close conformational resemblance to active porcine pancreatic elastase; its specificity pocket is buried, displaying the second "zymogen triad". Its five N-terminal residues are disordered, although the close activation site is fixed to the molecular surface. The structure of this native zymogen displays large conformational differences when compared with the recently solved crystal structure of bovine subunit III, an N-terminally truncated, non-activatable, proproteinase E variant lacking the first 13 residues of the native 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 surface-located and not involved in intra or inter-trimer contacts. This organization confers a functional role to the oligomeric structure, establishing 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 interactions, account for the high stability of the native ternary complex.
authors.affiliation: Institut de Biologia Fonamental i Departament de Bioquímica i BiologiaMolecular, Universitat Autònoma de Barcelona, Bellaterra, Spain. xgrcri@cid.csic.es
authors.forename: F X
authors.initials: FX
authors.lastname: Gomis-Rüth
authors.forename: M
authors.initials: M
authors.lastname: Gómez-Ortiz
authors.forename: J
authors.initials: J
authors.lastname: Vendrell
authors.forename: S
authors.initials: S
authors.lastname: Ventura
authors.forename: W
authors.initials: W
authors.lastname: Bode
authors.forename: R
authors.initials: R
authors.lastname: Huber
authors.forename: F X
authors.initials: FX
authors.lastname: Avilés
mesh_terms: D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry; D043422:Carboxypeptidases A; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry; D002919:Chymotrypsinogen / Q000737:chemistry; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000737:chemistry; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry*; D011487:Protein Conformation; D012697:Serine Endopeptidases; D013381:Subtilisins / Q000737:chemistry
chemical_list: D004792:Enzyme Precursors; D010446:Peptide Fragments; C057107:prosubtilisin; D002919:Chymotrypsinogen; D002268:Carboxypeptidases; D010450:Endopeptidases; D010447:Peptide Hydrolases; C035786:pro-proteinase E; D043422:Carboxypeptidases A; D012697:Serine Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; D008666:Metalloendopeptidases