Kinetic, Thermodynamic, and Structural Characterizations of the Association between Nrf2-DLGex Degron and Keap1

Abstract

cTranscription factor Nrf2 (NF-E2-related factor 2) coordinately regulates cytoprotective gene expression, but under unstressed conditions, Nrf2 is degraded rapidly through Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination. 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. In this study, we determined the minimum Keap1-binding sequence of the DLG motif, the low-affinity latch site, and defined a new DLGex motif that covers a sequence much longer than that previously defined. We have successfully clarified the crystal structure of the Keap1-DC-DLGex complex at 1.6 Å. DLGex possesses a complicated helix structure, which interprets well the human-cancer-derived loss-of-function mutations in DLGex. In thermodynamic analyses, Keap1-DLGex binding is characterized as enthalpy and entropy driven, while Keap1-ETGE binding is characterized as purely enthalpy driven. In kinetic analyses, Keap1-DLGex binding follows a fast-association and fast-dissociation model, while Keap1-ETGE binding contains a slow-reaction step that leads to a stable conformation. These results demonstrate that the mode of DLGex binding to Keap1 is distinct from that of ETGE structurally, thermodynamically, and kinetically and support our contention that the DLGex motif serves as a converter transmitting environmental stress to Nrf2 induction as the latch site.

Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response

“…An important finding for the Cys273 and Cys288 case is that these cysteine residues are in the IVR domain, which is located near the Kelch-DGR domain, so that the modifications of these residues by class II chemicals likely affects Keap1 binding to Nrf2 and ceases the ubiquitination of Nrf2. This model has been referred to as the "hinge and latch" mechanism and has been firmly supported by wide-ranging observations (37)(38)(39)(40). For instance, the IVR domain was found to surround the Kelch domain in our electron microscope analysis (37), and the proximity between the IVR and Kelch-DGR domains supports the notion that the modification of Cys273 and Cys288 is conveyed as a conformational distortion to the Kelch domain where Nrf2 binds (38,39).…”

“…For instance, the IVR domain was found to surround the Kelch domain in our electron microscope analysis (37), and the proximity between the IVR and Kelch-DGR domains supports the notion that the modification of Cys273 and Cys288 is conveyed as a conformational distortion to the Kelch domain where Nrf2 binds (38,39). Further structural, kinetic, and thermodynamic analyses of the Keap1-Nrf2 interaction (39), as well as somatic mutations analyses of Keap1 and Nrf2 in human cancers (40), also provide convincing lines of evidence that support this model. Thus, our conclusion for the consequence of Cys273 and Cys288 modification is that the conformational change in Keap1 elicited by the cysteine modification disrupts or distorts the Keap1-Nrf2 interaction and results in Nrf2 stabilization.…”

“…This model has been referred to as the "hinge and latch" mechanism and has been firmly supported by wide-ranging observations (37)(38)(39)(40). For instance, the IVR domain was found to surround the Kelch domain in our electron microscope analysis (37), and the proximity between the IVR and Kelch-DGR domains supports the notion that the modification of Cys273 and Cys288 is conveyed as a conformational distortion to the Kelch domain where Nrf2 binds (38,39). Further structural, kinetic, and thermodynamic analyses of the Keap1-Nrf2 interaction (39), as well as somatic mutations analyses of Keap1 and Nrf2 in human cancers (40), also provide convincing lines of evidence that support this model.…”

Absolute Amounts and Status of the Nrf2-Keap1-Cul3 Complex within Cells

“…6ϫHis-tagged proteins of Nrf2, Keap1, and Cul3 NTD were expressed in bacteria and purified as described previously (18,19,28,29). All of the proteins were purified using His-Trap FF crude (GE Healthcare) and were further purified with MonoQ or Superdex 200 column chromatography (GE Healthcare).…”

Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism

“…When cells are exposed to either electrophilic toxic chemicals or reactive oxygen species, the cysteine residues of Keap1 are modified, leading to the loss of Keap1 ubiquitin ligase activity. Consequently, Nrf2 is stabilized, accumulates in the nucleus (11)(12)(13)(14)(15)(16), and heterodimerizes with small Maf proteins to activate the transcription of cytoprotective enzymes and bind antioxidant response elements (AREs) and electrophile response elements (EpREs) (17).…”