The trimerization domain of nemo is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains
The trimerization domain of nemo is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains
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DOI：
10.1074/jbc.m314278200
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发表时间：
2004-07-02
影响因子：
4.8
通讯作者：
Véron, M
中科院分区：
文献类型：
--
作者：
Agou, F;Traincard, F;Véron, M
NEMO(NF-kappaB Essential Modator)作为IkappaB激酶(IKK)复合体的骨架/调节成分，在典型的NF-kappaB途径中起着关键作用。NEMO的自结合涉及多肽链的C-末端一半，包含两个假定的卷曲基序(CC2和LZ亮氨酸拉链)、一个富含Pro的区域和一个ZF锌指基序。使用纯化的截断突变体，我们证明了Nemo的最小寡聚化结构域是CC2-LZ片段，并且CC2和LZ亚结构域都是恢复Nemo缺陷细胞系中依赖于内毒素的NF-kappaB途径激活所必需的。我们证实了低聚结构域在三聚体中的结合，并研究了CC2和LZ亚结构域在低聚物构建中的特定作用。重组CC2-LZ多肽自结合成三聚体，结合常数接近野生型蛋白，而分离的CC2和LZ多肽分别形成结合常数较小的三聚体和二聚体。凝胶过滤和荧光各向异性实验表明，分离的CC2和LZ多肽混合后形成稳定的杂六角体。我们提出了一个激活的NEMO寡聚结构域的组织结构模型，其中三个C-末端结构域结合成一个伪六聚体，形成一个六螺旋束。这一模型与上游激活剂激活IKK复合体的机制有关。
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 wildtype 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.