1. Volume 21, Issue 5, Pages 689-700 (March 2006)
Structural Basis for Defects of Keap1 Activity Provoked by Its Point Mutations in Lung Cancer 
Balasundaram Padmanabhan, Kit I. Tong, Tsutomu Ohta, Yoshihiro Nakamura, Maria Scharlock, Makiko Ohtsuji, Moon-Il Kang, Akira Kobayashi, Shigeyuki Yokoyama, Masayuki Yamamoto 
Molecular Cell 
Volume 21, Issue 5, Pages (March 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 The Overall Tertiary Structure of mKeap1-DC
(A) Schematic diagram of Keap1 showing its BTB, IVR, DGR, and CTR domains, and Nrf2 showing its six domains, Neh1–Neh6.
(B and C) Ribbon representation of the mKeap1-DC β propeller domain is shown in top view as stereo (B) and in side view (C). Each β propeller blade is numbered from 1 to 6 and each β strand from I to IV.
(D) Locations of the two point mutations in human lung cancer cells (G364C and G430C) are shown in the ribbon structure.
(E) Structure-based sequence alignment of the six DGR repeats and CTR of mKeap1-DC. The positions of the four conserved β strands are both boxed in the sequence and indicated above the sequence by arrows. Each DGR repeat consists of four β strands with the exception of the first DGR repeat that lacks the strand β1. Conserved amino acid residues, such as double glycines (GG), arginine (R), tyrosine (Y), and tryptophan (W), are written in red. The positions of the two mutated glycines in human lung cancer cells are written in blue.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 The Structure-Function Relationship of the CTR
(A) Luciferase reporter assays performed in NIH3T3 cells with a series of alanine substitution Keap1 mutants. Keap1 harboring mutations in CTR is indicated schematically below the bar chart. The bar graph presents the mean ± SEM (n = 3).
(B) Subcellular localization of alanine-substituted Keap1 mutants. NIH3T3 cells were cotransfected with expression plasmids of Neh2-GFP and wild-type mKeap1 or its alanine mutants. Subcellular localization of Neh2-GFP was monitored by GFP (first row, Neh2-GFP). The Neh2-GFP fusion protein lacks a nuclear localization signal and therefore localizes in both the cytoplasm and nucleus of cells in the absence of Keap1 (Mock). Keap1 is shown by staining with anti-Keap1 antibody and anti-rabbit IgG conjugated with TRITC (second row, Keap1). The third row (Merge) shows the merged images of Neh2-GFP and mKeap1. Cells that show overlapping fluorescence of both mKeap1 and Neh2-GFP (double positives) are set as 100%. GVAV (30%) shows cells with cytoplasmic colocalization of mKeap1 and Neh2-GFP, which are 30% of the total double positives. GVAV (70%) shows cells with simultaneous cytoplasmic and nuclear localization of Neh2-GFP, which are 70% of the total double positives. The bottom panels (Nucleus) show images of DAPI nuclear staining.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Electrostatic Surface Potential of mKeap1-DC
(A–C) Presentation of the surface of mKeap1-DC, showing acidic, basic, and neutral residues in red, blue, and white, respectively, with side (A), top (B), and bottom (C) views.
(D) Basic patches on the bottom surface of mKeap1-DC are formed by a number of arginines and histidines, most of them highly conserved among human, rat, and mouse Keap1 molecules.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Structure of mKeap1-DC Complexed with the Neh2 Peptide
(A) Stereo view of the refined peptide (Leu-76–Leu-84) in the protein bound form. The final electron density 2mFo − DFc map is contoured at 1.0 σ.
(B) Side view of the surface of mKeap1-DC in complex with Neh2. Acidic, basic, and neutral residues are shown in red, blue, and white, respectively. Note that the Neh2 peptide, shown as yellow sticks, binds to a basic patch at the entrance of the central cavity on the bottom side of mKeap1-DC.
(C) Bottom view of (B).
(D) Stick representation showing the potential electrostatic interaction between Glu-79 of the Neh2 peptide and the residues of mKeap1-DC. The Neh2 peptide (light blue), the residues of mKeap1-DC (pink), and nitrogen (dark blue) and oxygen (red) atoms are shown. Electrostatic bonding is indicated by red dotted lines.
(E) Potential electrostatic interaction of Glu-82 of the Neh2 peptide with the residues of mKeap1-DC. A structural water molecule is depicted as a red circle. The color code of residues is as in (D). Gly-364 of Keap1 mutated in human lung cancer cell lines is located next to Ser-363, which is involved in interactions with Glu-82 of Nrf2 either directly or through a structural water molecule.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Somatic Mutation (G430C) and Gene Variation (G364C) in Keap1 Hamper Repression Activity of Keap1 on Nrf2
(A) Schematic diagram of hKeap1 showing its DGR and CTR domains. Asterisks indicate the two human lung cancer mutations G364C and G430C.
(B) Repression activity of the Keap1 mutants was monitored by a luciferase reporter assay. Wild-type or mutant Keap1 cDNA was transfected into Cos7 cells with Nrf2 cDNA and pRBGP2 luciferase reporter. Data represent mean ± SEM (n = 4) and transfection with Nrf2 cDNA alone served as a control.
(C) Association affinity to Nrf2 is reduced in the Keap1 mutants. 293T cells were transfected with expression plasmids for Nrf2 and FLAG-tagged wild-type Keap1 or the Keap1 mutants G364C or G430C. Protein complexes of Keap1 or its mutants were immunoprecipitated (IP) from whole-cell extracts with anti-FLAG antibody beads and immunoblotted (IB) with anti-Nrf2 antibody (top). The expression level of Nrf2 and FLAG-tagged Keap1 or mutants was verified by immunoblot analyses using anti-Nrf2 and anti-Keap1 antibodies (middle and bottom, respectively).
(D) The G364C and G430C Keap1 mutants are incapable of sequestering Nrf2 in the cytoplasm. Nrf2-GFP was expressed in Cos7 cells with wild-type Keap1 or mutants, and cells were stained with anti-FLAG antibody and anti-mouse IgG antibody conjugated with TRITC. Fluorescent views of Nrf2-GFP (first row) and Keap1 (TRITC, second row) are shown. Superposed views are shown as Merge.
(E) Bar chart presentation of the subcellular localization of Nrf2. A cell population with comparable fluorescent intensity in the nucleus and cytoplasm (C = N) is shown in white, with higher fluorescent intensity in the cytoplasm than in the nucleus in gray (C > N), and with higher fluorescent intensity in the nucleus in black (C < N).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Nrf2-Mediated Expression of Cytoprotective Genes Is Elevated in the Lung Cancer Cell Line Carrying KEAP1 Mutation
(A) Microarray analysis of cell lines derived from small cell lung carcinoma cases. Of the six cell lines, NCI-H1184 contains homozygous G364C mutation but the other cell lines do not harbor any mutations in the coding region of KEAP1 gene. The bar graph shows the average of duplicate runs on the expression of NQO1. The NCI accession numbers for these cell lines are as indicated.
(B) Knockdown experiment of the NRF2 gene expression in the lung adenocarcinoma cell line NCI-H1648, which also harbors homozygous G364C mutation in the KEAP1 gene. siRNA for the NRF2 gene (N) or scrambled control (C) is transfected. Numbers on the top of each panel show PCR cycles. GAPDH is the expression control.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
G364C and G430C Mutations Lead to Disruptions of the Intermolecular Interactions
(A) Close-up stereo view of the vicinity of Gly-430 and the intra- and intermolecular electrostatic interactions among the neighboring residues. Ser-431, which is located next to Gly-430, secures the position of Arg-415 at the interface through bonding with its adjacent residue, Asn-414.
(B) A cartoon to describe the detrimental effects of the human lung cancer somatic mutation (G430C) and gene variation (G364C) on the intermolecular association between mKeap1-DC and the Neh2 peptide.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions