Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

6A/05G267

Structural Basis for the Nrf2 interaction with the Keap1 repressor
Balasundaram Padmanabhan1*, Kit I.Tong2, Yoshihiro Nakamura1, Akira Kobayashi2, Masayuki
Yamamoto2 and Shigeyuki Yokoyama1,3,4 *1

RIKEN Genomic Sciences Center, Yokohama 230-0045, 2Graduate School of Comprehensive
Human Sciences, TARA and JST-ERATO Environmental Response Project, University of Tsukuba,
3Tsukuba 305-8577, Cellular Signaling Laboratory and Structurome Research Group, RIKEN Harima
Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, and 4Department ofBiophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo
113-0033, Japan
Introduction
Oxidative and electrophilic stresses provoke
physiological responses that induce the expression ofvarious cytoprotective genes. Recently, the transcription
factor Nrf2 was identified as the major regulator of thecytoprotective genes encoding phase 2 detoxication andantioxidant enzymes. Nrf2, which is normally localized
in the cytoplasm, tranlocates into nuclei when cells areexposed to electrophiles. Under normal conditions, thecytoplasmic Keap1 protein directly interacts with Nrf2
and negatively regulates the Nrf2 function, andpromoting its rapid degradation by the ubiquitindependent proteolytic pathway. Keap1 contains an Nterminal BTB domain, a conserved linker domain, and aC-terminal Kelch domain. The Kelch domain of Keap1
binds to the Neh2 domain of Nrf2, and enables Keap1
to sequester Nrf2 in the cytoplasm. To understand thefunctional importance of the Keap1 interaction for Nrf2,
we have determined and analyzed the crystal structureof the Kelch domain of Keap1 (Keap1-DC) as well asthat of the Keap1-DC complex with a Nrf2 interacting
peptide (Padmanabhan et al, 2005, 2006).
Materials and Methods
Mouse Keap1-DC was expressed in Escherichia coli
and purified, and crystallized as described elsewhere(Padmanabhan et al., 2005). For co-crystallization ofthe protein with the Neh2 peptide (LDEETGEFL), thepeptide was purchased from Promega K.K. The peptide
and protein solutions were mixed and incubated at 4°C
for about 3 hours before setting up the crystallization
experiment. The diffraction data for native and heavyatom derivatives were collected at the beamline BL26Bof SPring-8, and for the complex at BL6A of Photon
Factory, Japan. The structure was determined by theMIR method and refined to 1.60 Å with data from anative crystal. The current refined model at a resolutionof 1.60 Å consists of 290 residues, 8 sulfate ions, andFigure1. The overall tertiary structure of mKeap1-DC. Stereo ribbon representation of the mKeap1-DC β-propeller
domain is shown in “top view”. Each β-propeller blade is numbered from 1 to 6.

Users' Report227-1Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

346 water molecules, with a final R-factor of 16.8% andan Rfree of 20.0%. The electron density for 15 residues
at the N-terminus and 11 residues at the C-terminus wasabsent. For the protein-peptide complex, the initialorientation of the protein structure obtained from thenative crystal was optimized by rigid-body refinementin the respective crystal form. After maximum
likelihood refinement with CNS (Brunger et al., 1998),
the 2mFo-DFc and mFo-DFc maps were computed toidentify and build the peptide model.
Results and Discussion
The overall tertiary structure of mKeap1-DC isapproximately 49 Å x 36 Å, with a small
cylindrical/disc shaped structure, and consists of atandem of six twisted four-stranded (β1–β4) antiparallel
β-sheets resembling a 6-bladed β-propeller structurewith pseudo six-fold symmetry (Fig.1). In each kelch
module, the outermost fourth strand runs almost

perpendicular to the innermost first strand, which lies
nearly parallel to the central axis of the central cavity ofthe β-propeller. Like other β-propeller proteins, βstrands of both termini meet and, the disc-like
conformation is tightened by a “3+1” arrangement of βstrands. The peptide containing the ETGE motif binds
to bottom surface of the Keap1 DGR domain which
possesses highly basic surface-charged patch (Fig. 2).
The peptide possesses a tight four-residue β-hairpin
conformation comprised of the residues Asp-77, Glu-78,Glu-79, Thr-80, Gly-81, and Glu-82 (Fig. 2A). Our
mutational and affinity studies using isothermal
calorimetry (ITC) also demonstrated that the arginines,
such as Arg-415, at the rim of the central cavity on thebottom side of Keap1-DC and the ETGE motif in Neh2
are important for the inter-molecular interactions. Theseresults thus uncover the structural basis of the binding
interface between Keap1-DC and the Neh2 domain ofNrf2.

Figure2. The structure of mKeap1-DC complexed with a Neh2 peptide (Leu-76 to Leu-84). (A) Stereo view of therefined peptide in the protein bound form. The final electron density 2mFo-DFc map is contoured at 1.0 σ. (B)
Representation of the bottom view of the surface of mKeap1-DC in complex with Neh2. Acidic, basic, and neutral
residues are shown in red, blue, and white, respectively. The Neh2 peptide, shown as yellow sticks, binds to a basic patchat the entrance of the central cavity on the bottom side of mKeap1-DC. (C) Side view of (B).
Reference[1] Padmanabhan, B., Scharlock, M., Tong, K.I.,
Nakamura, Y., Kang, M.-I., Kobayashi, A., Matsumoto,T., Tanaka, A., Yamamoto, M. and Yokoyama, S. Acta
Cryst., F61 (2005) 153.
[2] Padmanabhan, B., Tong, K. I., Ohta, T., Nakamura,Y., Scharlock, M., Kang, M.-I., Kobayashi, A.,
Yokoyama, S. and Yamamoto, M. Molecular Cell 21(2006) 689.

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Users' Report*paddy@gsc.riken.jp
tokyo.ac.jp

oryokoyama@biochem.s.u-