Dissecting KEAP1-NRF2 interactions: Structural and functional impact of cancer-associated KEAP1 mutants

Abstract

Kelch-like ECH-associated protein 1 (KEAP1) is a cysteine-rich substrate adaptor for the CUL3-RBX1 E3 ubiquitin ligase complex, primarily regulating the transcription factor NRF2. By promoting NRF2 degradation under basal conditions, KEAP1 maintains redox homeostasis, while oxidative or electrophilic stress disrupts this regulation, enabling NRF2 activation of cytoprotective genes. Dysregulation of the KEAP1-NRF2 pathway, often through KEAP1 mutations, is implicated in cancer, where constitutive NRF2 activation promotes tumour growth, chemoresistance, and metabolic reprogramming. In this study, we investigated structural and functional consequences of three representative KEAP1 mutants R470C (superbinder), G480W (hypomorphic), and R554Q (null allele) using thermal shift assays (TSA), microscale thermophoresis (MST), isothermal calorimetry (ITC), and X-ray crystallography, complemented by AlphaFold3 modelling. TSA revealed that while inhibitor-induced thermal stability shifts followed similar pattern across mutants, baseline stability varied significantly, with R554Q showing the largest destabilisation. MST and ITC confirmed mutant-specific alterations in NRF2 ETGE peptide binding affinity, consistent with their known functional classifications. Structures of mutant-inhibitor and mutant-peptide complexes showed remarkable structural similarity to native KEAP1, with preserved inhibitor and peptide binding orientations despite residue substitutions. Spatial mapping of reported KEAP1 mutations revealed clustering of superbinder mutations near the IVR interface, while most loss-of-function variants localised within the KELCH domain. Our findings provide a detailed structural and biophysical characterization of clinically relevant KEAP1 mutations, offering mechanistic insights into their functional diversity. This work advances the understanding of KEAP1 mutation effects, with implications for therapeutic targeting of the KEAP1-NRF2 pathway in cancer.