Inter-domain distance analysis reveals conformational changes in the Keap1-Nrf2 stress sensor system.

The Keap1-Nrf2 system plays a central role in cellular defense against oxidative stress. Structural information on full-length Keap1 is essential for understanding the molecular basis of this regulation. However, its overall architecture has remained elusive due to pronounced conformational flexibility. In this study, we performed single-particle cryo-electron microscopy (cryo-EM) analysis of full-length Keap1 and found multiple particle conformations accompanied by severe preferred orientation in vitrified ice. To address these problems, we developed an analytical system that focuses on measuring the inter-domain distance between the two DC domains of the Keap1 homodimer. Using this approach, we identified the change of inter-domain distance distributions of Keap1 induced by Nrf2 binding or its inhibition, suggesting that these conformational changes are associated with Nrf2 regulation. Furthermore, through integration of this system with Keap1 deletion mutants, three flexible regions within Keap1 are found to contribute substantially to the conformational flexibility of the Keap1 homodimer. Together, these findings provide structural insights into the dynamic changes of the Keap1-Nrf2 system that would contribute to the development of Keap1-targeted therapeutics.