Integrative Modeling and Biochemical Analysis of the H ₂ S–Keap1–Cul3 Mechanism That Stabilizes Nrf2 and Promotes Ferroptosis Resistance

ABSTRACT

Ferroptosis is an iron‐dependent lipid‐peroxidation program with few mechanism‐based suppression strategies. Hydrogen sulfide (H ₂ S), an endogenous gasotransmitter, may restrain ferroptosis, but the structural route linking H ₂ S‐responsive Keap1 chemistry to Nrf2 stabilization is unresolved. We combined network pharmacology, Keap1–Cul3 structural modeling, three independent 100‐ns molecular dynamics (MD) simulations per state, exploratory molecular mechanics generalized Born surface area (MMGBSA) energy estimation, biochemical analysis of purified wild‐type and Cys151→Ser (C151S) Keap1, and cell‐based western blotting and quantitative PCR. Trajectories were assessed by root‐mean‐square deviation (RMSD), radius of gyration (Rg), and root‐mean‐square fluctuation (RMSF). Network analysis prioritized NFE2L2, HMOX1, AKT1 and SIRT1. Across replicates, the persulfidated state showed higher Cys151, Cul3‐binding‐region, and Cul3 RMSD with unchanged Rg, while Bric‐a‐brac/Tramtrack/Broad‐complex (BTB) self‐RMSD was lower than wild type, indicating altered BTB–Cul3 docking, not BTB destabilization. Interface contacts expanded from 20 to 45 residues, and the Glu192–Lys68 salt bridge was lost. In purified protein, NaHS generated a Cys151‐dependent persulfide without altering secondary structure, thermal stability, or BTB dimerization, reduced Cul3 affinity (dissociation constant: 0.9 ± 0.2–2.4 ± 0.5 μM) and lowered reconstituted Nrf2 ubiquitination to 40% of control; both effects were largely lost in C151S. In HepG2 cells, NaHS increased Nrf2, HMOX1, and phospho‐Akt/Akt and restored GPX4 under erastin‐induced stress. Persulfidation of Keap1 Cys151 weakens productive Cul3 engagement and reduces Nrf2 ubiquitination in vitro, with simulations indicating associated remodeling of the BTB–Cul3 interface, supporting H ₂ S‐responsive Keap1 modification as a contributor to Nrf2 stabilization and Akt/Nrf2/HMOX1–GPX4 anti‐ferroptotic defense.