Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains Toshihiko Oguraa, Kit I. Tongb, Kazuhiro Mioa, Yuusuke Maruyamaa, Hirofumi Kurokawab, Chikara Satoa,1, and Masayuki Yamamotob,2 aNeuroscience Research Institute and Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Umezono, Tsukuba 305-8568, Japan; and bDepartment of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan Communicated by Paul Talalay, Johns Hopkins University School of Medicine, Baltimore, MD, December 14, 2009 (received for review September 21, 2009) Keap1 is a substrate adaptor of a Cullin 3-based E3 ubiquitin ligase tin ligase complex that contributes to the rapid degradation of complex that recognizes Nrf2, and also acts as a cellular sensor for Nrf2 (19–22). Oxidative and electrophilic stresses covalently xenobiotics and oxidative stresses. Nrf2 is a transcriptional factor modify cysteine thiol residues of Keap1 and abrogate the regulating the expression of cytoprotective enzyme genes in re- Keap1-E3 ubiquitin ligase activity, which leads to the stabilization sponse to such stresses. Under unstressed conditions Keap1 binds and activation of Nrf2 (23). Thus, in response to such stress, Nrf2 Nrf2 and results in rapid degradation of Nrf2 through the protea- is released from Keap1 repression, accumulates in the nucleus, some pathway. In contrast, upon exposure to oxidative and elec- and activates transcription of cytoprotective enzyme genes. trophilic stress, reactive cysteine residues in intervening region Keap1 retains four discrete domains: i.e., BTB (Broad com- (IVR) and Broad complex, Tramtrack, and Bric-à-Brac domains of plex, Tramtrack and Bric-à-Brac), IVR (intervening region), Keap1 are modified by electrophiles. This modification prevents DGR (double glycine repeat), and CTR (C-terminal region). Nrf2 from rapid degradation and induces Nrf2 activity by repres- The DGR and CTR domains in collaboration form a β-propeller sion of Keap1. Here we report the structure of mouse Keap1 homo- structure and interact with the Neh2 domain of Nrf2 (hereafter dimer by single particle electron microscopy. Three-dimensional we refer to these two domains of Keap1 as the DC domain). The reconstruction at 24-Å resolution revealed two large spheres at- IVR domain contains two critical cysteine residues, Cys272 and tached by short linker arms to the sides of a small forked-stem Cys288, that are important for repression of Nrf2 activity. This structure, resembling a cherry-bob. Each sphere has a tunnel corre- suggests that these residues are important for the electrophilic sponding to the central hole of the β-propeller domain, as deter- stress sensing (23, 24). Whereas the BTB domain acts to dimerize mined by x-ray crystallography. The IVR domain appears to Keap1, the Cys151 residue in the BTB domain also appears to be β surround the core of the -propeller domain. The unexpected prox- important in stress sensing, even though it is not required for the β imity of IVR to the -propeller domain suggests that any distortions repression of Nrf2 activity per se. generated during modification of reactive cysteine residues in the We describe a unique molecular mechanism employed in the β IVR domain may send a derepression signal to the -propeller repression of Nrf2 activity. The substrate adaptor Keap1 forms a domain and thereby stabilize Nrf2. This study thus provides a struc- dimer to accommodate Nrf2 and the stoichiometry of Nrf2: tural basis for the two-site binding and hinge-latch model of stress Keap1 is 1∶2. So that the Keap1 dimer recognizes and binds sensing by the Nrf2-Keap1 system. two evolutionarily conserved degrons, DLG and ETGE in Nrf2. These two motifs show two orders of magnitude difference eap1 (Kelch-like ECH-associated protein 1) is a multifunc- in the association constant to the DC domain of Keap1. The two- Ktional protein that represses activity of the transcriptional site binding enables Keap1 to initiate efficient ubiquitination of factor Nrf2 (NF-E2-related Factor 2). Nrf2 promotes expression Nrf2 (25–27). Importantly, perturbation of the Keap1-Nrf2 bind- of various cytoprotective genes in response to xenobiotic and ing, especially at the weak DLG site, blocks Nrf2 degradation – oxidative insults (1 3). Whereas Keap1-null mutant mice are (a hinge and latch mechanism) and this constitutes the critical elec- juvenile-lethal due to hyperkeratosis of the esophagus (4), hepa- trophilic/oxidative stress sensor function (28, 29). The necessity of tocyte-specific knockout of the Keap1 gene robustly elevates both degrons for efficient Nrf2 turnover has been supported accumulation of Nrf2 in the nucleus and protects hepatocytes through structural, biochemical, and clinical analyses (27, 30). against acute drug toxicity and inflammatory liver injury (5, 6). To understand the molecular mechanisms underlying the inter- In contrast, the risk of oxidant-induced acute lung injury is sig- action of Keap1 with Nrf2, two groups of investigators have nificantly increased upon dysfunction of the Nrf2 gene (7). Simi- solved the crystal structure of the substrate-binding DC domain larly, Nrf2-null mutant mice are susceptible to environmental of mouse and human Keap1 (15, 31). Nonetheless, the molecular toxicants and various pathologies, including hepatotoxicity, pneumotoxicity, neurotoxicity, carcinogenicity, and inflammation (8–10). The induction of Phase 2 enzymes through augmentation Author contributions: T.O., K.I.T., K.M., H.K., C.S., and M.Y. designed research; T.O., K.I.T., of the Nrf2 activity has been widely accepted as a promising ap- K.M., and C.S. performed research; T.O., Y.M., and C.S. analyzed data; T.O., K.I.T., H.K., C.S., proach for cancer chemoprevention, for protection against and M.Y. wrote the paper. oxidative stress, and for chemoprophylaxis against stress-related The authors declare no conflict of interest. disorders (11–14). Indeed, a number of Keap1 loss-of-function Data deposition: The EM reconstruction volume has been deposited in the Electron somatic mutations have been identified in human lung cancers Microscopy Data Bank, www.emdatabank.org (EMD ID code EMD-1675). 1 and cancer-derived cell lines in which Nrf2 is constitutively active To whom correspondence may be addressed at: Department of Medical Biochemistry, – Tohoku University Graduate School of Medicine, 2-1 Seinyo-cho, Aoba-ku, Sendai (15 17). The latter finding suggests that Keap1 may sensitize 980-8575, Japan. E-mail: [email protected] cancer cells to chemotherapy. 2To whom correspondence may be addressed. E-mail: [email protected]. Keap1 is a member of newly described BTB and C-terminal ac.jp Kelch (BACK) domain-containing protein family (18). Keap1 This article contains supporting information online at www.pnas.org/cgi/content/full/ is a substrate adaptor component of a Cul3-dependent E3 ubiqui- 0914036107/DCSupplemental. 2842–2847 ∣ PNAS ∣ February 16, 2010 ∣ vol. 107 ∣ no. 7 www.pnas.org/cgi/doi/10.1073/pnas.0914036107 Downloaded by guest on September 25, 2021 mechanism whereby modification of IVR Cys residues affects the binding of Nrf2 degron motifs to the DC domain remains to be clarified. This is partly due to the difficulty in crystallizing the full-length dimeric Keap1 protein. To this end, we examined the three-dimensional imaging/reconstruction analysis of mouse Keap1 homodimer by EM by use of negatively stained proteins. Results Purification of Mouse Keap1 Protein. Recombinant mouse Keap1 (M1—R614) protein with a C-terminal six-His tag was purified by Ni-NTA agarose affinity chromatography and twice by Super- dex S-200 size exclusion chromatography (SEC). Keap1 was eluted from the second SEC as a sharp peak at 1.03 mL (Fig. 1A), which corresponds to a major protein band with an apparent mo- lecular weight of approximately 80 kDa in SDS-PAGE (Fig. 1B). The major protein band was confirmed to be Keap1 by immuno- blotting analysis with anti-Keap1 antibody (Fig. 1C, right). A faint band at 150 kDa corresponds to the size of Keap1 dimer. An aliquot at 1.03 ml elution was used for the EM study. Molecular Shape of Keap1 by EM. Mouse Keap1 recombinant pro- tein was negatively stained and imaged by EM at x 52; 100. Keap1 displayed variously shaped particles of uniform size with dimeric assembly (Fig. 2A). Most particles had two isolated spherical bodies joined via linkers to a forked-stem structure (Fig. 2B). The distance between the two spherical masses varied, reflecting different orientations of the same molecule. Although rarely BIOCHEMISTRY Fig. 2. EM image of negatively stained Keap1. (A) Keap1 particles were ob- served as uniformly sized projections (arrowheads). Protein is shown in bright shades. Scale bar, 200 Å. (B) Examples of the Keap1 projections with sche- matic diagrams below each panel. An example of a minor population with extremely distant round spheres is also displayed at the rightmost bottom. Scale bar, 100 Å. For statistical analysis, 216 particles were automatically picked up by the auto-accumulation method and utilized as training data for the three-layer neural network (NN) auto-picking system. The trained NN selected 12,651 particles for analysis. observed, the image of two spheres joined by a straight linker (Fig. 2B, Upper Left) is postulated to be the top view, while par- ticles with a hinged linker likely represent side views (Fig. 2B, Lower Second from Right). Minor projections with extremely dis- tant spherical bodies were also observed (Fig. 2B, Lower Right). Three-Dimensional Reconstruction of Keap1. We then performed a three-dimensional reconstruction (32) using our Single Particle Image analysis method using Neural Networks and Simulated an- nealing programs (33–36) and the IMAGIC V software (37). From the initial 12,651 EM images, 9,827 particles (77.7%) were Fig.