Hydrogen peroxide (H 2O 2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H 2O 2. One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H 2O 2. In this study, we present crystallographic evidence for the H 2O 2-sensing mechanism and H 2O 2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H 2O 2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H 2O 2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H 2O 2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H 2O 2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H 2O 2induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H 2O 2.
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - 11 Dec 2018|
Bibliographical noteCopyright © 2018 the Author(s). Published by PNAS.
- Amino Acid Substitution
- Bacterial Proteins/chemistry
- Binding Sites/genetics
- Corynebacterium glutamicum/genetics
- Crystallography, X-Ray
- Genes, Bacterial
- Hydrogen Peroxide/metabolism
- Mutagenesis, Site-Directed
- Protein Structure, Quaternary
- Transcription Factors/chemistry
- Transcription, Genetic