Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling

Abstract

Keap1 is a BTB-Kelch substrate adaptor protein that regulates steady-state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. We have determined the structure of the Kelch domain of Keap1 bound to a 16-mer peptide from Nrf2 containing a highly conserved DxETGE motif. The Nrf2 peptide contains two short antiparallel beta-strands connected by two overlapping type I beta-turns stabilized by the aspartate and threonine residues. The beta-turn region fits into a binding pocket on the top face of the Kelch domain and the glutamate residues form multiple hydrogen bonds with highly conserved residues in Keap1. Mutagenesis experiments confirmed the role of individual amino acids for binding of Nrf2 to Keap1 and for Keap1-mediated repression of Nrf2-dependent gene expression. Our results provide a detailed picture of how a BTB-Kelch substrate adaptor protein binds to its cognate substrate and will enable the rational design of novel chemopreventive agents.

Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy

“…X-ray crystal structures of the Keap1-DC-DLG peptide and Keap1-DC-ETGE-peptide complexes have depicted that the side chains of Q26, D27, and D29 of the DLG motif ( 23 LxxQDxDLG 31 ) and E79 and E82 of the ETGE motif ( 77 DxETGE 82 ) have direct interactions with the Keap1-DC. Whereas T80 has both inter-and intramolecular interactions, Q75 has intramolecular contact (14,21,22). Cancer-related mutations within the ETGE motif are primarily substitutions of these essential residues for Keap1 recognition whereas mutations in the DLG motif are either changes in the conserved (D29 and L30) or nonconserved residues (W24, I28, V32, and R34).…”

“…2 A-C), compromised the association with Keap1-DC. In par- Using the x-ray crystal structure of human Keap1-DC in association with the ETGE peptide as template (PDB code 2FLU) (22) and by means of the MODELLER 9v1 program (23), we modeled some of the amino acid substitutions found in cancer patients or cell lines to access their impact on the structurefunction relationship of Keap1 with Nrf2. The model detailing the structure of the E79Q mutation (Figs.…”

Cross-cancer profiling of molecular alterations within the human autophagy interaction network

“…Overexpression of SQSTM1/p62 sets up a positive feedback loop that further activates NFE2L2 through competitive binding of the NFE2L2 inhibitor KEAP1. Three of 4 HNSC-G2 patients mutant for NFE2L2 harbored previously characterized E79 or E82 activating changes 21 and we found median SQSTM1/P62 mRNA levels to be higher in HNSC NFE2L2 mutants versus nonmutants ( Table 2), suggesting that increased SQSTM1/p62-mediated autophagic degradation of KEAP1 could occur in NFE2L2 mutant HNSC patients with poor OS.…”

“…2): CDKN2A, 11,12 KEAP1, 11 NFE2L2/ NRF2, 11-13 and MTOR. 13,14 Within cancer types, UCEC had the highest number of significantly mutated AA genes, including: PPP2R1A and BIRC6 (F > 0.1); LRRK2, TAB3, CDK5RAP2, RABGAP1, and RB1CC1 (F D 0.05-0.1) ; TBC1D15, ULK4, MTMR7, WDR45, DNM1L, MAPK8, PRKACG, TRAPPC8, ATG4C, RAB24, CDKN2AIP, HACE1, KBTBD7, PRKAA2, VTI1B, ARF6, 21 were observed in multiple cancers (HNSC, LUSC, UCEC); similarly, convergent hotspot mutations were present in CDKN2A 15 (W110* and E120*) in both HNSC and LUSC, which are squamous cell carcinomas of distinct tissue type that share mutational landscapes. 11 The low frequency of mutation of core autophagy genes suggests that the autophagy machinery is functional in patients of the 11 cancer types investigated, and therefore remains exploitable by these tumor types.…”

“…KEAP1 mutations predicted to be LOF (Table S1) were found in both subtypes, while NFE2L2 mutations were found only in LUSC, as well as in HNSC and UCEC. Several LUSC and HNSC patients had previously characterized E79 NFE2L2 activating mutations, 21 while activating E82 mutations were identified only in UCEC patients (Table S1). SQSTM1 is a target gene for transcriptional upregulation by NFE2L2, 77 and KEAP1 mutants in both LUAD and LUSC had significantly higher SQSTM1 mRNA levels compared to wild-type patients ( Table 2; Table S5); therefore, as previously suggested, a positive-feedback loop may exist in patients with overactive NFE2L2/NRF2 (through NFE2L2 and/or KEAP1 mutation), whereby transcriptionally upregulated levels of SQSTM1 further stabilize NFE2L2 protein levels, through increased SQSTM1-mediated autophagic degradation of KEAP1.…”