Neuroscience Research Institute and Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-4 Umezono, Tsukuba 305-8568, Japan.
Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2842-7. doi: 10.1073/pnas.0914036107. Epub 2010 Jan 27.
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-à-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-A 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.
Keap1 是一种 Cullin 3 基 E3 泛素连接酶复合物的底物衔接子，可识别 Nrf2，并且还充当细胞对外源物和氧化应激的传感器。Nrf2 是一种转录因子，可调节细胞保护酶基因的表达，以应对这些应激。在未受应激的条件下，Keap1 与 Nrf2 结合，导致 Nrf2 通过蛋白酶体途径快速降解。相比之下，当暴露于氧化和亲电应激时，Keap1 的 intervening region (IVR) 和 Broad complex、Tramtrack、Bric-à-Brac 结构域中的反应性半胱氨酸残基被亲电物质修饰。这种修饰阻止了 Nrf2 的快速降解，并通过抑制 Keap1 诱导 Nrf2 活性。在这里，我们通过单颗粒电子显微镜报告了小鼠 Keap1 同源二聚体的结构。在 24-A 分辨率下的三维重建揭示了两个大球体通过短连接臂附着在小叉形茎结构的两侧，类似于樱桃状。每个球体都有一个与β-三叶螺旋结构域中心孔相对应的隧道，这是通过 X 射线晶体学确定的。IVR 结构域似乎围绕着β-三叶螺旋结构域的核心。IVR 结构域与β-三叶螺旋结构域的意外接近表明，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-三叶螺旋结构域发送去抑制信号，从而稳定 Nrf2。这项研究为 Nrf2-Keap1 系统的双位点结合和铰链闩锁模型提供了结构基础。
