Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation.

Alexandre Appolaire 1 Eva Rosenbaum 1 M Asunción Durá 1 Matteo Colombo 1 Vincent Marty 1 Marjolaine Noirclerc-Savoye 1 Anne Godfroy 2 Guy Schoehn 1 Eric Girard 1 Frank Gabel 1 Bruno Franzetti 1
1 IBS - UMR 5075 - Institut de biologie structurale
2 LM2E - Laboratoire de microbiologie des environnements extrêmophiles
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.
Keywords : REVEALS BLEOMYCIN HYDROLASE TRICORN PROTEASE RAY SOLUTION SCATTERING SMALL-ANGLE SCATTERING CRYSTAL-STRUCTURE PROTEIN-DEGRADATION ASPARTYL AMINOPEPTIDASE LEUCINE AMINOPEPTIDASE SUBSTRATE-SPECIFICITY
Type de document :
Article dans une revue
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2013, 288 (31), pp.22542-54. 〈10.1074/jbc.M113.450189〉
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Soumis le : vendredi 18 juillet 2014 - 09:32:24
Dernière modification le : lundi 19 février 2018 - 14:34:03

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Alexandre Appolaire, Eva Rosenbaum, M Asunción Durá, Matteo Colombo, Vincent Marty, et al.. Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2013, 288 (31), pp.22542-54. 〈10.1074/jbc.M113.450189〉. 〈hal-01025541〉

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