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

Abstract

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 α-helix followed by a mini antiparallel β-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 β-propeller structure of Keap1. In contrast, the central α-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 β-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 α-helix in a correct position to benefit ubiquitin signaling.

We thank Kouhei Tsumoto, Midori Oinuma, and Rieko Ishima for advice on interpreting the ITC data, for instruction on the use of the ITC instrument, and for discussion of the NMR data, respectively. We also thank Balasundaram Padmanabhan for the gift of the ETGE peptide and Akira Kobayashi for discussions. We thank Tania O'Connor for critical reading of the manuscript.

This work is supported in part by grants-in-aid from JST-ERATO, the Ministry of Education, Culture, Sports, Science, and Technology. T.T. is supported by the 21st Century COE Program and the Protein 3000 project.