Abstact

The KEAP1-CUL3 ubiquitin ligase regulates protein stability of transcriptional factor NRF2 and plays critical roles in cellular stress response. The BTB domain of KEAP1 functions as a sensor for electrophilic chemicals. However, the precise mechanisms by which electrophiles are recognized and inhibit BTB activity remain unclear. Here, we show that electrophilic modification alters the spatial arrangement of the BTB homodimer, regulating its ligase activity. Co-crystal structural analyses and functional studies using potent NRF2-inducing CDDO-derivatives, synthetic electrophilic compounds structurally related to clinically approved molecules such as Omaveloxolone, revealed that the key sensor residue, Cys151, resides in a structurally elaborate environment within the BTB domain. Modification of Cys151 by NRF2 inducers changes the spatial configuration of the CUL3-binding sites in the BTB homodimer, reducing KEAP1-CUL3 complex affinity. In contrast, a Cys151-targeting NRF2 inhibitor induces an opposite rearrangement of the BTB homodimer. This study elucidates the molecular mechanism by which the BTB domain finely regulates KEAP1-CUL3 ubiquitin ligase activity.