crystallization papers 2346 doi:10.1107/S0907444904024825 Acta Cryst. (2004). D60, 2346±2348 Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Crystallization and initial crystallographic analysis of the Kelch domain from human Keap1 Xuchu Li, a Donna Zhang, b Mark Hannink a,b and Lesa J. Beamer a * a Department of Biochemistry, University of Missouri±Columbia, Columbia, MO 65211, USA, and b Life Sciences Center, University of Missouri±Columbia, Columbia, MO 65211, USA Correspondence e-mail: beamerl@missouri.edu # 2004 International Union of Crystallography Printed in Denmark ± all rights reserved 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. Received 3 July 2004 Accepted 1 October 2004 1. Introduction A major mechanism for protection against oxidative damage in eukaryotic cells involves the coordinated induction of a group of cyto- protective genes including the glutathione- S-transferases, NAD(P)H oxidoreductase and  -glutamylcysteine synthetase (Dalton et al., 1999; Nguyen et al., 2003). Induction of these cytoprotective genes during conditions of oxidative stress enables the neutralization of chemically reactive molecules and the restoration of cellular redox homeostasis. These genes share common cis-acting DNA sequences termed antioxidant-response elements (AREs) that mediate transcriptional induction following exposure to oxidative stress (Dalton et al., 1999; Nguyen et al., 2003; Wasserman & Fahl, 1997). The transcription factor Nrf2 has emerged as the critical regulator of ARE-dependent tran- scription (Nguyen et al., 2003; Jaiswal, 2004). Nrf2 is a member of a small family of tran- scription factors that share a conserved bZIP- dimerization/DNA-binding domain and the ability to bind ARE-like DNA-sequence motifs (Nguyen et al., 2003; Motohashi et al., 2002). The major regulator of Nrf2 is a BTB- Kelch protein termed Keap1, which regulates both the subcellular localization and steady- state levels of Nrf2 (Wakabayashi et al., 2003; Itoh et al., 1999; Zhang & Hannink, 2003). Recent work from our laboratory has demon- strated that Keap1 targets Nrf2 for ubiquiti- nation in the context of a Cul3-dependent E3 ubiquitin ligase complex (Zhang et al., 2004). Exposure of cells to reactive chemicals or oxidative stress enables Nrf2 to escape Keap1- mediated repression, leading to increased transcription of Nrf2-dependent genes (Zhang & Hannink, 2003). Keap1 is one of more than 50 human proteins that share an N-terminal BTB domain, a central linker domain and a C-terminal Kelch domain. BTB-Kelch proteins are generally regarded as actin-binding proteins with diverse biological roles in the regulation of the cytoskeleton (Adams et al. , 2000). For example, a number of single point mutations within the GAN1 gene have been described that result in giant axonal neuro- pathy, an autosomal recessive disease char- acterized by defects in intermediate ®lament organization in sensorimotor neurons (Bomont et al., 2000). The crystal structures of several mammalian BTB domains have been reported, including the human PLZF transcription factor and the Skp1 protein, which bridges the Cul1 protein and numerous F-box substrate-adaptor proteins in the SCF1 E3 ubiquitin ligase complexes (Ahmad et al., 1998; Wu et al., 2003). The high degree of homology between different BTB domains has allowed the construction of homology-based models that