Crystallization and initial crystallographic analysis of the Kelch domain from human Keap1.
The human Keap1 protein is a substrate adaptor for an E3 ubiquitin ligase complex that specifically 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 beta-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 resolution under cryocooling conditions. A selenomethionine-substituted version of the Kelch domain has also been purified 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.
Li
Xuchu
X
Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA.
Zhang
Donna
D
Hannink
Mark
M
Beamer
Lesa J
LJ
D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D005692:Galactose Oxidase / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D000072019:Kelch-Like ECH-Associated Protein 1; D018384:Oxidative Stress; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D014965:X-Rays
D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D011506:Proteins; D005692:Galactose Oxidase