title: Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation.
abstract: Tetrahedral (TET) aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle x-ray scattering revealed that it is a dimer whose architecture in solution is identical to that determined by x-ray crystallography within the fully assembled TET particle. Small angle x-ray scattering also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild-type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis, and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo.
authors.affiliation: Institut de Biologie Structurale, CNRS, UMR5075, F-38027/Commissariat à l'Energie Atomique, F-38054/Université Joseph Fourier, F-38027 Grenoble, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Appolaire
authors.forename: Eva
authors.initials: E
authors.lastname: Rosenbaum
authors.forename: M Asunción
authors.initials: MA
authors.lastname: Durá
authors.forename: Matteo
authors.initials: M
authors.lastname: Colombo
authors.forename: Vincent
authors.initials: V
authors.lastname: Marty
authors.forename: Marjolaine Noirclerc
authors.initials: MN
authors.lastname: Savoye
authors.forename: Anne
authors.initials: A
authors.lastname: Godfroy
authors.forename: Guy
authors.initials: G
authors.lastname: Schoehn
authors.forename: Eric
authors.initials: E
authors.lastname: Girard
authors.forename: Frank
authors.initials: F
authors.lastname: Gabel
authors.forename: Bruno
authors.initials: B
authors.lastname: Franzetti
mesh_terms: D003001:Cloning, Molecular; D019281:Dimerization; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D043902:Pyrococcus horikoshii / Q000201:enzymology*
chemical_list: D010447:Peptide Hydrolases
keywords: Aminopeptidase; Archaea; Biophysics; Peptidases; Proteasome; Protein Degradation; Protein Self-assembly; Proteolytic Enzymes; Structural Biology; X-ray Scattering