Structure of the BTB Domain of Keap1 and Its Interaction with the Triterpenoid Antagonist CDDO

Abstract

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

The Keap1–Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases

“…The β1 helix is essential for the formation of the dimeric interface. The N-terminal residues form the domainswapped β-sheet, which also plays a key role in the homodimerisation interface formation (Cleasby et al 2014).…”

“…Among the cysteine residues, Cys151, Cys171, Cys273 and Cys288 are highly reactive, which are present in the BTB-IVR domains of Keap1 (Cleasby et al 2014) (Fig. 2b).…”

“…2b) on all three domains of Keap1 may provide an alternative solution to the inhibition of Keap1. The cysteine residues (C151, C273 and C288) on the BTB-IVR domain decrease the binding of CUL3 to the former and, thus, the Nrf2 degradation pathway is affected (Cleasby et al 2014). However, Nrf2 is not released substantially for nuclear import.…”

“…In such cases, overexpression of Nrf2 helps in the induction of the ARE pathway. The triterpenoid CDDO adducts covalently bonded with C151 of the BTB domain and ensures that there is no interaction of the CUL3 with the Keap1 (Cleasby et al 2014) (Fig. 6).…”

“…6). The intervening region (IVR) of Keap1 consists of the cysteine residues C273 and C288 that attract electrophilic binding and, hence, cause distortion of the structural fold that leads to improper positioning of the protein domains and, hence, disrupt the binding of the degradation complex required for its regular functioning (Cleasby et al 2014). …”