Structural Characterization of KEAP1 R415K to Guide the Design of KEAP1‐Nrf2 Peptide Inhibitors

KEAP1型 氧化应激 化学 转录因子 对接(动物) 结合位点 生物化学 氧化磷酸化 基因 医学 护理部
作者
Gwendolyn Pyeatt,Samantha N. Muellers,Adrian Whitty,Karen N. Allen
出处
期刊:The FASEB Journal [Wiley]
卷期号:35 (S1)
标识
DOI:10.1096/fasebj.2021.35.s1.04626
摘要

Oxidative stress can cause extensive damage to DNA, proteins, and lipids. Thus, long-term oxidative stress has been linked to many diseases including cancer and Alzheimer's disease. The highly-charged binding interface between regulatory protein Kelch-like ECH-associated protein 1 (KEAP1) and transcription factor nuclear erythroid factor 2-like 2 (Nrf2) is a common drug target for diseases related to oxidative stress. In the absence of oxidative stress, KEAP1 binds with high affinity to the DxETGE motif of Nrf2. Oxidative stress causes KEAP1 to release Nrf2, allowing it to translocate to the nucleus and upregulate the transcription of antioxidant enzymes. Our current peptide inhibitors of this protein-protein interaction (PPI) are modeled after the DxETGE motif and have exhibited low cell permeability, prompting the bioisosteric replacement of charged residues. We previously identified Arg415 as the KEAP1 residue with the most significant contribution to binding energy at the KEAP1-Nrf2 interface. It has been observed that the R415A mutation abolishes Nrf2 binding, and so a more conservative KEAP1 R415K variant was characterized to better understand the interactions between this residue and residue Glu79 on Nrf2. The variant was characterized using X-ray crystallography, differential scanning fluorimetry, and the binding was assessed using a competitive fluorescence anisotropy binding assay. This mutation caused a 40-fold decrease in binding affinity, which suggested that retaining the charge alone was not sufficient to restore wild-type binding affinity. KEAP1 R415K was successfully crystallized in 8% w/v polyethylene glycol 20,000, 0.2 M imidazole, and 0.01 M nickel (II) chloride hexahydrate, and the crystals diffracted to a resolution of 2.43 Å. Structures of the unliganded R415K Kelch domain were determined using molecular replacement. Optimization of the crystals toward the structure determination of the R415K variant bound to peptide inhibitors is underway. These liganded structures can then be used to assess the binding interactions between KEAP1 Arg415 and Nrf2 Glu79 and guide future peptide inhibitor optimizations to improve their potency and bioavailability.

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