Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO

PLoS One. 2014 Jun 4;9(6):e98896. doi: 10.1371/journal.pone.0098896. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Humans
  • Imidazoles / chemistry*
  • Imidazoles / pharmacology
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
  • Intracellular Signaling Peptides and Proteins / chemistry*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Kelch-Like ECH-Associated Protein 1
  • Models, Molecular
  • Molecular Conformation
  • Mutation
  • Oleanolic Acid / analogs & derivatives*
  • Oleanolic Acid / chemistry
  • Oleanolic Acid / pharmacology
  • Protein Binding
  • Protein Interaction Domains and Motifs*
  • Structure-Activity Relationship

Substances

  • 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole
  • Imidazoles
  • Intracellular Signaling Peptides and Proteins
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • Oleanolic Acid

Grants and funding

The research described in this article was funded by GlaxoSmithKline and Astex Pharmaceuticals. The funders played a role in study design, data collection and analysis, decision to publish, and preparation of the manuscript.