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PMID: 16581765 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model.

Molecular and cellular biology ·Vol. 26 ·No. 8 ·2006-04-00 ·Pages 2887-900

Tong KI, Katoh Y, Kusunoki H, Itoh K, Tanaka T, Yamamoto M

Abstract

The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.

MeSH Terms
Adaptor Proteins, Signal Transducing/genetics,metabolism Amino Acid Motifs Amino Acid Sequence Animals Binding Sites Calorimetry Conserved Sequence Cytoskeletal Proteins/genetics,metabolism Escherichia coli/genetics Kelch-Like ECH-Associated Protein 1 Mice Models, Chemical Models, Molecular Molecular Sequence Data NF-E2-Related Factor 2/chemistry,genetics,isolation & purification,metabolism Nuclear Magnetic Resonance, Biomolecular Point Mutation Protein Binding Protein Processing, Post-Translational Protein Structure, Secondary Protein Structure, Tertiary Recombinant Fusion Proteins/chemistry,metabolism Sequence Homology, Amino Acid Thermodynamics Ultracentrifugation
Chemicals
Adaptor Proteins, Signal Transducing Cytoskeletal Proteins Keap1 protein, mouse Kelch-Like ECH-Associated Protein 1 NF-E2-Related Factor 2 Recombinant Fusion Proteins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Tong Kit I
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8572, Japan.
Katoh Yasutake
Kusunoki Hideki
Itoh Ken
Tanaka Toshiyuki
Yamamoto Masayuki
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.26.8.2887-2900.2006
Published
2006-04-00
Pages
2887-900
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC1446969
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.26.8.2887-2900.2006 PMC Full Text View journal details
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