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Molecular and Cellular Biology, April 2006, p. 2887-2900, Vol. 26, No. 8
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.8.2887-2900.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Keap1 Recruits Neh2 through Binding to ETGE and DLG Motifs: Characterization of the Two-Site Molecular Recognition Model

Kit I. Tong,1,2,4 Yasutake Katoh,1,2,4 Hideki Kusunoki,1 Ken Itoh,1,2,4 Toshiyuki Tanaka,1,3* and Masayuki Yamamoto1,2,4*

Center for Tsukuba Advanced Research Alliance,1 Graduate School of Comprehensive Human Sciences,2 Graduate School of Life and Environmental Sciences,3 JST-ERATO Environmental Response Project, University of Tsukuba, Tsukuba, Japan4

Received 29 April 2005/ Returned for modification 4 June 2005/ Accepted 21 January 2006

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central {alpha}-helix followed by a mini antiparallel ß-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the ß-propeller structure of Keap1. In contrast, the central {alpha}-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two ß-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central {alpha}-helix in a correct position to benefit ubiquitin signaling.

* Corresponding author. Mailing address for Toshiyuki Tanaka: Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8572, Japan. Phone: 81 29 853 6706. Fax: 81 29 853 6706. E-mail: ttanaka{at}tara.tsukuba.ac.jp. Mailing address for Masayuki Yamamoto: Center for TARA, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan. Phone: 81 29 853 6158. Fax: 81 29 853 7318. E-mail: masi{at}tara.tsukuba.ac.jp.

Molecular and Cellular Biology, April 2006, p. 2887-2900, Vol. 26, No. 8
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.8.2887-2900.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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