Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains
Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains
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DOI：
10.1073/pnas.0914036107
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发表时间：
2010-02-16
影响因子：
11.1
通讯作者：
Yamamoto, Masayuki
中科院分区：
文献类型：
--
作者：
Ogura, Toshihiko;Tong, Kit I.;Yamamoto, Masayuki
Keap1 是基于 Cullin 3 的 E3 泛素连接酶复合物的底物接头，可识别 Nrf2，并且还可充当外源物质和氧化应激的细胞传感器。 Nrf2 是一种转录因子，可调节细胞保护酶基因的表达以应对此类应激。在无应激条件下，Keap1 结合 Nrf2，并通过蛋白酶体途径导致 Nrf2 快速降解。相反，当暴露于氧化和亲电子应激时，Keap1 的介入区 (IVR) 和 Broad 复合体、Tramtrack 和 Bric-a-Brac 结构域中的反应性半胱氨酸残基会被亲电子试剂修饰。这种修饰可防止 Nrf2 快速降解，并通过抑制 Keap1 来诱导 Nrf2 活性。在这里，我们通过单粒子电子显微镜报告了小鼠 Keap1 同二聚体的结构。 24 埃分辨率的三维重建揭示了两个大球体通过短连接臂连接到一个小叉状茎结构的侧面，类似于樱桃鲍勃。根据 X 射线晶体学测定，每个球体都有一个与 β 螺旋桨域的中心孔相对应的隧道。 IVR 域似乎围绕着 beta-propeller 域的核心。 IVR 与 β-螺旋桨结构域出人意料的接近表明，在 IVR 结构域中反应性半胱氨酸残基修饰过程中产生的任何扭曲都可能向 β-螺旋桨结构域发送去抑制信号，从而稳定 Nrf2。因此，这项研究为 Nrf2-Keap1 系统应力传感的双位点结合和铰链锁模型提供了结构基础。
Keap1 is a substrate adaptor of a Cullin 3-based E3 ubiquitin ligase complex that recognizes Nrf2, and also acts as a cellular sensor for xenobiotics and oxidative stresses. Nrf2 is a transcriptional factor regulating the expression of cytoprotective enzyme genes in response to such stresses. Under unstressed conditions Keap1 binds Nrf2 and results in rapid degradation of Nrf2 through the proteasome pathway. In contrast, upon exposure to oxidative and electrophilic stress, reactive cysteine residues in intervening region (IVR) and Broad complex, Tramtrack, and Bric-a-Brac domains of Keap1 are modified by electrophiles. This modification prevents Nrf2 from rapid degradation and induces Nrf2 activity by repression of Keap1. Here we report the structure of mouse Keap1 homodimer by single particle electron microscopy. Three-dimensional reconstruction at 24-angstrom resolution revealed two large spheres attached by short linker arms to the sides of a small forked-stem structure, resembling a cherry-bob. Each sphere has a tunnel corresponding to the central hole of the beta-propeller domain, as determined by x-ray crystallography. The IVR domain appears to surround the core of the beta-propeller domain. The unexpected proximity of IVR to the beta-propeller domain suggests that any distortions generated during modification of reactive cysteine residues in the IVR domain may send a derepression signal to the beta-propeller domain and thereby stabilize Nrf2. This study thus provides a structural basis for the two-site binding and hinge-latch model of stress sensing by the Nrf2-Keap1 system.