KEAP1 C151 active site catalysis drives electrophilic signaling to upregulate cytoprotective enzyme expression

作者：Matthew R. Schnell, Tianhua Zhai, Edwin Ragwan, Hae-Chang Jung, Jiayu Zhang, Anthony F. Lagalante, Yan Kung, Daniel A. Kraut, Zuyi Huang, Aimee L. Eggler · 发表于：Redox Biology · 年份：2025 · DOI：10.1016/j.redox.2025.103906 · 被引用次数：13 · 研究领域：Genomics, phytochemicals, and oxidative stress、Plant-derived Lignans Synthesis and Bioactivity、Redox biology and oxidative stress

Cells mount a detoxification, antioxidant, and anti-inflammatory response to electrophiles, mediated by the NRF2 transcription factor. Electrophilic NRF2 activators are used to treat diverse chronic diseases. While the majority of NRF2 activators target C151 of KEAP1, the primary NRF2 repressor, it is unknown how diverse electrophiles favor this particular cellular cysteine. One hypothesis is that the p K a of C151 is lowered by surrounding basic residues, resulting in a higher population of the reactive thiolate. We show that the p K a of C151 is 6.9, providing optimal reactivity at physiological pH, using the fluorogenic, thiol-reactive electrophile monobromobimane. Surprisingly, monobromobimane reacts with C151 much faster than with a small-molecule thiolate. NRF2 activators in clinical use and trials (omaveloxolone, bardoxolone methyl, and sulforaphane) readily compete with monobromobimane for C151. A BTB-monobimane crystal structure shows no specific orientation in the active site after covalent addition. A 4D flexible BTB model based on seven crystal structures was used to dock mBBr and NRF2 activators into the active site to obtain poses of the pre-covalent enzyme-substrate complexes. They reveal an active site around C151 that accommodates structurally diverse activators using largely hydrophobic interactions, with a hydrogen bond orienting their electrophilic carbons within ∼3-5 Å of C151 for a catalytic proximity effect. In addition, our biochemical and docking resu...