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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.

SUBMITTER: Lo SC

PROVIDER: S-EPMC1538563 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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

Lo Shih-Ching SC Li Xuchu X Henzl Michael T MT Beamer Lesa J LJ Hannink Mark M

The EMBO journal 20060803 15

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 poc ...[more]

PMID: 16888629

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Project description:Under oxidative and electrophilic stresses, cells launch an NRF2-mediated transcriptional antioxidant program. The activation of NRF2 depends on a redox sensor, KEAP1, which promotes the ubiquitination and degradation of NRF2. While a great deal has been learned about this duo, its quantitative signaling properties are largely unexplored. Here we examined these properties, including half-life, maximal activation, and response steepness (ultrasensitivity) of NRF2, through mathematical modeling. The models describe the binding of KEAP1 and NRF2 via ETGE and DLG motifs, NRF2 production, KEAP1-dependent and independent NRF2 degradation, and perturbations by different classes of NRF2 activators. Simulations revealed at the basal condition, NRF2 is sequestered by KEAP1 and the KEAP1-NRF2 complex is distributed comparably in an ETGE-bound (open) state and an ETGE and DLG dual-bound (closed) state. When two-step ETGE binding is considered, class I-V, electrophilic NRF2 activators shift the balance to a closed state incompetent to degrade NRF2, while the open and closed KEAP1-NRF2 complexes transition from operating in cycle mode to equilibrium mode. Ultrasensitive NRF2 activation (a steep rise of free NRF2) can occur when NRF2 nearly saturates KEAP1. The ultrasensitivity results from zero-order degradation through DLG binding and protein sequestration through ETGE binding. Optimal abundances of cytosolic and nuclear KEAP1 exist to maximize ultrasensitivity. These response characteristics do not require disruption of DLG binding as suggested by the hinge-latch hypothesis. In comparison, class VI NRF2 activators cause a shift to the open KEAP1-NRF2 complex and ultimately its complete dissociation, resulting in a fast release of NRF2 followed by stabilization. However, ultrasensitivity is lost due to decreasing free KEAP1 abundance. In summary, by simulating the dual role of KEAP1, i.e., sequestering and promoting degradation of NRF2, our modeling provides novel quantitative insights into NRF2 activation, which may help design novel NRF2 modulators and understand the oxidative actions of environmental stressors.

Dataset Information

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

Publications

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

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