Abstract
The Keap1-Nrf2 system is central for mammalian cytoprotection against various stresses and a drug target for disease prevention and treatment. One model for the molecular mechanisms leading to Nrf2 activation is the Hinge-Latch model, where the DLGex-binding motif of Nrf2 dissociates from Keap1 as a latch, while the ETGE motif remains attached to Keap1 as a hinge. To overcome the technical difficulties in examining the binding status of the two motifs during protein-protein interaction (PPI) simultaneously, we utilized NMR spectroscopy titration experiments. Our results revealed that latch dissociation is triggered by low-molecular-weight Keap1-Nrf2 PPI inhibitors and occurs during p62-mediated Nrf2 activation, but not by electrophilic Nrf2 inducers. This study demonstrates that Keap1 utilizes a unique Hinge-Latch mechanism for Nrf2 activation upon challenge by non-electrophilic PPI-inhibiting stimuli, and provides critical insight for the pharmacological development of next-generation Nrf2 activators targeting the Keap1-Nrf2 PPI.
Highlights
The Keap1-Nrf[2] system is central for mammalian cytoprotection against various stresses and a drug target for disease prevention and treatment
Our results unequivocally demonstrate that Keap1–Nrf[2] protein-protein interaction (PPI) inhibitors and p62, but not electrophilic cysteine-targeting compounds, utilize the Hinge-Latch mechanism for Nrf[2] activation
We have addressed this issue by utilizing titration nuclear magnetic resonance (NMR) spectroscopy, as the unique mode of interaction between Keap[1] and Nrf[2] allows us to apply this technique for the two-site PPI analysis
Summary
The Keap1-Nrf[2] system is central for mammalian cytoprotection against various stresses and a drug target for disease prevention and treatment. Whereas the DLGex motif possesses a three-helix structure and binds weakly to Keap1-DC (Double glycine repeat or Kelch, plus C-terminal) domain, the ETGE motif is a single β-hairpin structure that binds tightly to a pocket in Keap1-DC domain in a key-and-lock manner[8,9]. The binding of these two motifs of Nrf[2] to the Keap[1] homodimer (i.e., two-site binding) is strictly required for ubiquitination and proteasomal degradation of Nrf[27,10]. These mutations disrupt the two-site binding of Keap1–Nrf[2] and lead to constitutive accumulation of Nrf[2], supporting malignant growth of cancer cells[14]
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