Keap1 recruits Neh2 through binding to ETGE and DLG motifs: Characterization of the two-site molecular recognition model

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.