FC0076
NF-E2-related factor 2 (Nrf2)  -  Keap1

Biological function
Transcription factor Nrf2 (NF-E2-related factor 2) plays important roles in the cellular defense against various electrophilic and oxidative stresses. Keap1 (Kelch-like ECH-associated protein 1) negatively controls Nrf2 signaling. Nrf2 coordinately regulates cytoprotective gene expression, but under unstressed conditions, Nrf2 is degraded rapidly through Keap1 (Kelch-like ECH- associated protein 1)-mediated ubiquitination. Nrf2 is rapidly ubiquitinated by Keap1-Cul3 (Cullin 3) E3 ligase.

Structural evidence
Nrf2 beyond residue 39 is invisible in the structure of the complex.

Biochemical evidence
The DLG motif is much longer than the previously defined ’classical’ DLG motif; thus, it is also referred as the extended DLG motif as the DLGex motif, which contains both the classical DLG motif and the DIDLID element. Deletion analysis shows that region spanning Met17 to Tyr46 is essential and sufficient for Keap1 binding. Met17 to Gln51 retains slightly stronger Keap1- binding activity. Met17 to Gln51 as the extended DLG motif of Nrf2 (Nrf2-DLGex). The DLGex peptide generates 14 potential intermolecular electrostatic interactions between Keap1 and the DC domain. (k_a was 6.1 x 10⁴ M^-1 s^-1) and dissociation rate constant (k_d was 0.196 s^-1), it is concluded that the binding of Nrf2-DLGex to Keap1-DC has fast association and dissociation modes. Nrf2 harbors two Keap1-binding motifs, DLG and ETGE. Interactions between these two motifs and Keap1 constitute a key regulatory nexus for cellular Nrf2 activity through the formation of a two-site binding hinge-and-latch mechanism. DLG motif is the low-affinity latch site, Keap1-DLGex binding is characterized as enthalpy and entropy driven, while Keap1-ETGE binding is characterized as purely enthalpy driven. DLGex binding to Keap1 is distinct from that of ETGE structurally, thermodynamically, and kinetically. The DLGex motif serves as a converter transmitting environmental stress to Nrf2 induction as the latch site.

Structure/Mechanism
One Nrf2 molecule associates with a Keap1 homodimer or two Keap1 molecules by exploiting two distinct binding motifs within the Neh2 domain, the low- affinity DLG motif and the high-affinity ETGE motif. This two- site binding of Nrf2 to the Keap1 homodimer allows for the efficient ubiquitination of Nrf2, which leads to the rapid degradation of Nrf2 through the proteasome pathway. There are seven lysine residues between the DLG and ETGE motifs; thus, the two- site binding of Nrf2 to Keap1 is assumed to be critical for efficient Nrf2 ubiquitination. This rapid turnover of Nrf2 maintains a constitutively low cellular Nrf2 level. In contrast, upon exposure to toxic electrophiles, the electrophiles modify reactive cysteine residues of Keap1 and disrupt the two-site binding process by altering the conformation of the Keap1 homodimer. Consequently, Nrf2 is no longer degraded and accumulates in the nucleus, where it activates the expression of cytoprotective genes. The low- affinity DLG site is dissociated from Keap1 upon exposure to electrophiles have been accumulating.

Mechanism category
tethering

Significance
Fuzziness underlies the low-affinity DLG and Keap1 interaction, which serves as a converter transmitting electrophilic insults to Nrf2 induction. The DLGex motif consists of efficient sensor machinery acting as a latch that easily dissociates from Keap1-DC upon detecting electrophilic and oxidative stress.

Medical relevance
Human KEAP1 and NRF2 have also been identified in various types of cancers. Importantly, all somatic mutations in NRF2 re- side near the two degron sites, the DLG and ETGE motifs.