title: The trimerization domain of NEMO is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains.
abstract: NEMO (NF-kappaB essential modulator) plays a key role in the canonical NF-kappaB pathway as the scaffold/regulatory component of the IkappaB kinase (IKK) complex. The self-association of NEMO involves the C-terminal halves of the polypeptide chains containing two putative coiled-coil motifs (a CC2 and a LZ leucine zipper), a proline-rich region, and a ZF zinc finger motif. Using purified truncation mutants, we showed that the minimal oligomerization domain of NEMO is the CC2-LZ segment and that both CC2 and LZ subdomains are necessary to restore the LPS-dependent activation of the NF-kappaB pathway in a NEMO-deficient cell line. We confirmed the association of the oligomerization domain in a trimer and investigated the specific role of CC2 and LZ subdomains in the building of the oligomer. Whereas a recombinant CC2-LZ polypeptide self-associated into a trimer with an association constant close to that of the wild-type protein, the isolated CC2 and LZ peptides, respectively, formed trimers and dimers with weaker association constants. Upon mixing, isolated CC2 and LZ peptides associated to form a stable hetero-hexamer as shown by gel filtration and fluorescence anisotropy experiments. We propose a structural model for the organization of the oligomerization domain of activated NEMO in which three C-terminal domains associate into a pseudo-hexamer forming a six-helix bundle. This model is discussed in relation to the mechanism of activation of the IKK complex by upstream activators.
authors.affiliation: Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 2185, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. fagou@pasteur.fr
authors.forename: Fabrice
authors.initials: F
authors.lastname: Agou
authors.forename: François
authors.initials: F
authors.lastname: Traincard
authors.forename: Emilie
authors.initials: E
authors.lastname: Vinolo
authors.forename: Gilles
authors.initials: G
authors.lastname: Courtois
authors.forename: Shoji
authors.initials: S
authors.lastname: Yamaoka
authors.forename: Alain
authors.initials: A
authors.lastname: Israël
authors.forename: Michel
authors.initials: M
authors.lastname: Véron
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016880:Anisotropy; D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D051550:I-kappa B Kinase; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection; D016335:Zinc Fingers
chemical_list: D018076:DNA, Complementary; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010455:Peptides; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse