The Trimerization Domain of Nemo Is Composed of the Interacting C-terminal CC2 and LZ Coiled-coil Subdomains

• Published in: The Journal of biological chemistry Vol. 279; no. 27; pp. 27861 - 27869
• Main Authors: Agou, Fabrice, Traincard, François, Vinolo, Emilie, Courtois, Gilles, Yamaoka, Shoji, Israël, Alain, Véron, Michel
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 02.07.2004
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Anisotropy; Cell Line; Chromatography; Chromatography, Gel; Dimerization; DNA, Complementary; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Genetic Complementation Test; Genetic Vectors; I-kappa B Kinase; Kinetics; Life Sciences; Lipopolysaccharides; Lipopolysaccharides - metabolism; Mice; Microscopy, Fluorescence; Models, Genetic; Molecular Sequence Data; Mutation; NF-kappa B; NF-kappa B - metabolism; Peptides; Peptides - chemistry; Precipitin Tests; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases; Protein-Serine-Threonine Kinases - chemistry; Recombinant Proteins; Recombinant Proteins - chemistry; Transfection; Zinc Fingers
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M314278200

NEMO (NF-κB essential modulator) plays a key role in the canonical NF-κB pathway as the scaffold/regulatory component of the IκB 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-κB 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.