Redox properties of the A‐domain of the HMGB1 protein

Abstract

The High Mobility Group B1 (HMGB1) protein plays important roles in both intracellular (reductive) and extracellular (oxidative) environments. We have carried out quantitative investigations of the redox chemistry involving Cys22 and Cys44 of the HMGB1 A-domain, which form an intramolecular disulfide bond. Using NMR spectroscopy, we analyzed the realtime kinetics of the redox reactions for the A-domain in glutathione and thioredoxin systems, and also determined the standard redox potential. Thermodynamic experiments showed that the Cys22-Cys44 disulfide bond stabilizes the folded state of the protein. These data suggest that the oxidized HMGB1 may accumulate even in cells under oxidative stress. Structured summary:MINT-6795963: txn (uniprotkb:P10599) and HMGB1 (uniprotkb:P09429) bind (MI:0408) by nuclear magnetic resonance (MI:0077)

Redox Modification of Cysteine Residues Regulates the Cytokine Activity of High Mobility Group Box-1 (HMGB1)

“…The structure:function relationship between modifications on these residues to the inflammatory activity of HMGB1 is unknown. The formation of an intramolecular disulfide bond between C23 and C45 stabilizes the folded state of the fulllength protein and generates a conformation change (9) that might impact upon the signaling function of HMGB1 as an inflammatory mediator (9,10). The results of our present study indicate that the inflammatory activities of HMGB1 require both reduced C106 and the formation of an intramolecular disulfide bond between C23 and C45.…”

“…These redox-dependent changes will thereby regulate many critical functions of signaling proteins including HMGB1 (18,19). C23 and C45 readily form a disulfide bond (20) which increases the stability of the folded full length HMGB1 molecule; NMR analysis confirms that this linkage alters the conformation of the A domain within the HMGB1 protein (9,18). C23 and C45 mutations prevent HMGB1 from binding to the intracellular target protein Beclin 1, stopping HMGB1 from initiating cellular autophagy processes (21).…”

Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy

“…No reducing regent was used in the column chromatography procedures except in the sonication buffer. Complete disulfide bond formation in HMGB1 was confirmed by NMR (21).…”

“…For all-thiol HMGB1 in the buffer, 5 mM DTT was also added, and the NMR tube was sealed in the presence of argon gas. Resonance assignment was also aided by previous assignment data for HMGB1 under different conditions (21,(33)(34)(35). NMR data were processed and analyzed using the NMRPipe (36) and NMRView (37) programs.…”

“…When released into the extracellular space, HMGB1 is initially in the reduced state ("all-thiol HMGB1") (see Fig. 1A) but becomes oxidized due to the oxidative environment (5,15,21). HMGB1 has three cysteine residues.…”