Keap1 is a BTB-Kelch substrate adaptor protein that regulates steady-state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. We have determined the structure of the Kelch domain of Keap1 bound to a 16-mer peptide from Nrf2 containing a highly conserved DxETGE motif. The Nrf2 peptide contains two short antiparallel beta-strands connected by two overlapping type I beta-turns stabilized by the aspartate and threonine residues. The beta-turn region fits into a binding pocket on the top face of the Kelch domain and the glutamate residues form multiple hydrogen bonds with highly conserved residues in Keap1. Mutagenesis experiments confirmed the role of individual amino acids for binding of Nrf2 to Keap1 and for Keap1-mediated repression of Nrf2-dependent gene expression. Our results provide a detailed picture of how a BTB-Kelch substrate adaptor protein binds to its cognate substrate and will enable the rational design of novel chemopreventive agents.

Keap1 is a substrate adaptor protein for an ubiquitin ligase complex that targets the Nrf2 transcription factor for degradation. Keap1 binds Nrf2 through its C-terminal Kelch domain, which contains six copies of the evolutionarily conserved kelch repeat sequence motif. The structure of the Kelch domain from human Keap1 has been determined by x-ray crystallography to a resolution of 1.85 Å. The Kelch domain forms a 6-bladed ␤-propeller structure, with residues at the C terminus forming the first strand in the first blade. Key structural roles have been identified for the highly conserved glycine, tyrosine, and tryptophan residues that define the kelch repeat sequence motif. In addition, we show that substitution of a single amino acid located within a loop that extends out from the bottom of the ␤-propeller structure abolishes binding of Nrf2. The structure of the Kelch domain of Keap1 represents a high quality model for the superfamily of eukaryotic kelch repeat proteins and provides insight into how disease-causing mutations perturb the structural integrity of the Kelch domain.

The Kelch repeat is a common sequence motif in eukaryotic genomes and is approximately 50 amino acids in length. The structure of the Kelch domain of the human Keap1 protein has previously been determined at 1.85 Angstrom, showing that each Kelch repeat forms one blade of a six-bladed beta-propeller. Here, use of 1.35 Angstrom SAD data for de novo structure determination of the Kelch domain and for refinement at atomic resolution is described. The high quality and resolution of the diffraction data and phase information allows a detailed analysis of the role of solvent in the structure of the Kelch repeat. Ten structurally conserved water molecules are identified in each blade of the Kelch beta-propeller. These appear to play distinct structural roles that include lining the central channel of the propeller, interacting with residues in loops between strands of the blade and making contacts with conserved residues in the Kelch repeat. Furthermore, we identify a conserved C-H...pi hydrogen bond between two key residues in the consensus Kelch repeat. This analysis extends our understanding of the structural roles of conserved residues in the Kelch repeat and highlights the potential role of solvent in maintaining the fold of this common eukaryotic structural motif.

The human Keap1 protein is a substrate adaptor for an E3 ubiquitin ligase complex that speci®cally targets the transcription factor Nrf2 for degradation. Keap1 functions as a sensor of oxidative stress, such that the inhibition of Keap1-dependent degradation of Nrf2 activates a genetic program that protects cells from reactive chemicals and maintains cellular redox homeostasis. Keap1 interacts with Nrf2 through its C-terminal Kelch-repeat domain. Kelch-repeat domains are found in a large number of proteins and are predicted to assemble into a -propeller structure. Only a single Kelch-repeat domain, that from the fungal enzyme galactose oxidase, has had its structure determined. Here, the crystallization of the Kelch domain of human Keap1 protein by hanging-drop vapor diffusion is reported in space group P6 5 22. Crystals diffract to 1.85 A Ê resolution under cryocooling conditions. A selenomethionine-substituted version of the Kelch domain has also been puri®ed and crystallizes isomorphously with the native protein. Structure determination by MAD phasing is under way. The role of Keap1 in oxidative stress and cytoprevention suggests that the Kelch domain will be an attractive target for therapeutic drug design.

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