Crystal structure of the kelch domain of human keap1

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Nrf2-Keap1 signal transduction pathway senses oxidative stress and protects eukaryotic cells against oxidative damage and cancer. The transcription factor Nrf2 induces Phase II stress response genes, which enable cells to neutralize reactive molecules and restore cellular redox homeostasis. Keap1 is a BTBKelch protein that regulates both the subcellular localization and steady state levels of Nrf2. Recently our lab has identified Keap1 as a redox-regulated substrate adaptor protein for a Cullin3-dependent E3 ubiquitin ligase complex, which targets Nrf2 for degradation in a controlled manner. The research project presented in this dissertation is to study the interaction between Nrf2 and Keap1 starting from a structural biology approach. The N-terminal Neh2 domain of Nrf2 mediates binding of Nrf2 to the Kelch domain of Keap1. We solved the crystal structure of the Kelch domain of human Keap1 and carried out detailed analysis of the structural/solvent features in this domain. This domain structure, which was the first high-resolution structure of a mammalian Kelch domain, demonstrated that the Kelch domain of Keap1 is a six-bladed ?-propeller that uses a C-terminal mode of closure. The structure revealed how conserved amino acids and water molecules contribute to both inter- and intra-blade stability and provided insight into how disease-causing mutations perturb the structural integrity of the Kelch domain. We found that the bottom loops of the Kelch domain are critical for its sufficient association with the Neh2 domain. Based on mutagenesis data, we mapped out a Neh2-Kelch interface on the bottom side of the Kelch domain. We further crystallized a putative Kelch-Neh2 complex that diffracts to 4[angstrom]. We are now in the process of obtaining high quality crystals of the Neh2-Kelch complex for structure determination purpose.--From public.pdf