Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.
Highlights
Reactive oxygen species (ROS) from the environment or generated by the cellular metabolism can cause oxidative damage to proteins, DNA, and lipids, leading to diseases such as cancer, dementia, and cardiovascular disease, to name a few [1,2]
FL-Nuclear factor erythroid 2-related factor 2 (Nrf2) is an acidic protein with pI ~4.8
The molecular weight of FL-Nrf2 is only 70.4 kDa, it runs with an apparent MW of ~110 kDa on SDS-PAGE gels as already reported previously [44] (Figure S1)
Summary
Reactive oxygen species (ROS) from the environment or generated by the cellular metabolism can cause oxidative damage to proteins, DNA, and lipids, leading to diseases such as cancer, dementia, and cardiovascular disease, to name a few [1,2]. In the presence of oxidative stress, several redox-sensitive cysteines in Keap (e.g., C151, C273, and C288) are modified by ROS, resulting in protein conformational changes and disruption of Nrf binding [31,32] This leads to the accumulation of Nrf in the nucleus and subsequent activation of ARE-dependent gene transcription. The solution structure of a 79-residue fragment (PDB: 2LZ1; residues 445–523 of human Nrf2) representing only part of the Neh domain (lacking the C-terminal leucine-zipper dimerization region) was solved using NMR spectroscopy [43]. Upon binding with Kelch, the DLG and ETGE binding motifs in the Neh domain of Nrf became protected, yet the rest of the protein remained highly dynamic These unique structural properties may be involved in regulating the interactions of Nrf with other proteins and determine its function in response to oxidative stress
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