NEMO trimerizes through its coiled-coil C-terminal domain
NEMO trimerizes through its coiled-coil C-terminal domain
复制标题
DOI：
10.1074/jbc.m201964200
复制
发表时间：
2002-05-17
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;Ye, F;Véron, M
Nemo/IkappaB激酶(IKK)γ是IKK复合体的调节成分，由两个蛋白激酶IKKalpha和IKKbeta组成。为了研究Nemo的自组装特性，进一步了解IKK复合体的激活机制，我们纯化了在大肠杆菌中表达的野生型和突变型Nemo。在没有IKK伴侣的情况下，重组NEMO(RNEMO)是一种亚稳态的功能单体，根据其荧光和远紫外圆二色谱正确折叠，它与IKK络合物特异结合。发现一小部分rNEMO与DNAK(E.coliHsp70)密切相关。我们还研究了Nemo与原核和真核Hsp70的相互作用，结果表明Hsp70-Nemo复合体形成了可能对应于组装中间体的超分子结构。体内交联实验表明，与IKK结合的天然NEMO处于二聚体和三聚体之间的平衡状态。与天然Nemo类似，从其ikk结合N-末端结构域(残基242-388)删除的Nemo突变体形成稳定的三聚螺旋线圈，这表明Nemo与ikk或Hsp70的结合防止了错误的域间配对反应，这些反应可能导致rNEMO的聚集或非天然低聚状态。我们提出了一个模型，在这个模型中，IKK复合体的激活是通过NEMO与一个尚未确定的上游激活剂结合后的三聚来发生的。
NEMO/IkappaB kinase (IKK) gamma is the regulatory component of the IKK complex comprising the two protein kinases, IKKalpha and IKKbeta. To investigate the self-assembly properties of NEMO and to understand further the mechanism of activation of the IKK complex, we purified wildtype and mutant NEMO expressed in Escherichia coli. In the absence of its IKK partners, recombinant NEMO (rNEMO) is a metastable functional monomer correctly folded, according to its fluorescence and far-UV CD spectra, which is binding specifically to the IKK complex. A minor fraction of rNEMO was found tightly associated with DnaK (E. coli Hsp70). We also examined the interaction of NEMO with prokaryotic and eukaryotic Hsp70, and we showed that the Hsp70-NEMO complex forms a supramolecular structure probably corresponding to an assembly intermediate. In vivo crosslinking experiments indicate that native NEMO in association with IKK is in equilibrium between a dimeric and a trimeric form. Similarly to native NEMO, a NEMO mutant deleted from its IKK binding N-terminal domain (residues 242-388) forms a stable trimeric coiled-coil, suggesting that the association of NEMO with IKK or with Hsp70 prevents incorrect interdomain pairing reactions that could lead to aggregation or to an non-native oligomeric state of rNEMO. We propose a model in which the activation of the IKK complex occurs through the trimerization of NEMO upon binding to a not yet identified upstream activator.