Cysteine thiol-to-sulfonate oxidation induces unfolding for the functional switching of the extracellular HMGB1 protein

Oxidation of cysteine thiols to sulfonate groups (–SO₃^–) by reactive oxygen species can regulate protein function. Near the end of inflammation, this modification in the extracellular HMGB1 protein abolishes its proinflammatory activity. Using NMR spectroscopy, we investigated how thiol-to-sulfonate oxidation switches HMGB1’s function. Our data show that the oxidation of cysteine 106 (C106) induces unfolding of the HMGB1 B-box domain. In contrast, other chemical modifications, such as S-glutathionylation, at the same cysteine did not have this effect, highlighting the unique impact of thiol-to-sulfonate oxidation. Employing ¹³C direct-detected NMR, we characterized the oxidized B-box domain. NMR data confirmed global unfolding but revealed residual α-helical propensity near the second and third helices. NMR paramagnetic relaxation enhancement data revealed electrostatic impacts of the C106 thiol-to-sulfonate oxidation. To test whether unfolding is driven by negative charge in a hydrophobic environment, we analyzed the C106D variant, as aspartate electrostatically mimics cysteine sulfonate. However, the C106D variant remained folded, even though NMR confirmed a negative charge at D106. Further NMR experiments showed that the –SO₃⁻ group at residue 106 drastically slows down the protein folding kinetics, compared with the –COO⁻ group at the same position, suggesting that –SO₃⁻ introduces a large desolvation penalty for protein folding. This study illuminates protein unfolding via thiol-to-sulfonate oxidation of a cysteine residue in a hydrophobic environment as a mechanism for protein functional switching. Since HMGB1 is a therapeutic target for inflammatory diseases, understanding this inactivation mechanism offers insight for designing covalent inhibitors.

This work was supported by Grant R35-GM130326 from the NIH (to J.I.) and Grant H-2104-20250403 from the Welch Foundation (to J.I.). We thank Dr. Tianzhi Wang for maintenance of the NMR equipment; Dr. Luis Holthauzen for assistance in the Circular dichroism experiments; and Dr. William Russell for the mass spectrometry analysis at the University of Texas Medical Branch Mass Spectrometry Core Facility (supported by the Cancer Prevention Research Institute of Texas Grant RP190682 and RP250644).