Different Electrostatic Potentials Define ETGE and DLG Motifs as Hinge and Latch in Oxidative Stress Response

Abstract

Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.

We are grateful to M. Kobayashi, H. Motohashi, and K. Itoh for helpful discussions. We are indebted to T. Tanaka for use of the isothermal calorimeter, Y. Nakamura for helping in data collection, and T. O'Connor for critical reading of the manuscript.

This work was supported in part by the RIKEN Structural Genomics/Proteomics Initiative; the National Project on Protein Structural and Functional Analyses; the Japanese Ministry of Education, Culture, Sports, Science and Technology; and ERATO-JST.