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Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path

Nature Structural & Molecular Biology volume 11, pages 1179–1185 (2004)Cite this article

Abstract

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s−1. The disulfide bond–mediated conformation switch results in a metastable form that is locally strained by ∼3 kcal mol−1. On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.

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Figure 1: MALDI-TOF spectra for the tryptic digests of reduced (top) and oxidized (bottom) wild-type OxyR.
Figure 2: AMS analysis of OxyR cysteines in cells treated with H2O2.
Figure 3: Characterization of OxyR oxidation by chemical modification.
Figure 4: Steady-state fluorescence spectra of OxyR.
Figure 5: Stopped-flow measurement of conformational switch of OxyR during oxidation.
Figure 6: Intermediates in the structural transitions of OxyR.
Figure 7: Urea-induced equilibrium unfolding transition of OxyR.

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References

  1. Halliwell, B. & Gutteridge, J.M.C. Free Radicals in Biology and Medicine Oxford University Press, New York (1999).

    Google Scholar 

  2. Lander, H.M. An essential role for free radicals and derived species in signal transduction. FASEB J. 11, 118–124 (1997).

    Article  CAS  Google Scholar 

  3. Storz, G., Tartaglia, L.A. & Ames, B.N. Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science 248, 189–194 (1990).

    Article  CAS  Google Scholar 

  4. Zheng, M., Aslund, F. & Storz, G. Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279, 1718–1721 (1998).

    Article  CAS  Google Scholar 

  5. Hausladen, A. et al. Nitrosative stress: activation of the transcription factor OxyR. Cell 86, 719–729 (1996).

    Article  CAS  Google Scholar 

  6. Kullik, I., Toledano, M.B., Tartaglia, L.A. & Storz, G. Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation. J. Bacteriol. 177, 1275–1284 (1995).

    Article  CAS  Google Scholar 

  7. Tao, K., Fujita, N. & Ishihama, A. Involvement of the RNA polymerase alpha subunit C-terminal region in co-operative interaction and transcriptional activation with OxyR protein. Mol. Microbiol. 7, 859–864 (1993).

    Article  CAS  Google Scholar 

  8. Toledano, M.B. et al. Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell 78, 897–909 (1994).

    Article  CAS  Google Scholar 

  9. Aslund, F., Zheng, M., Beckwith, J. & Storz, G. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc. Natl. Acad. Sci. USA 96, 6161–6165 (1999).

    Article  CAS  Google Scholar 

  10. Tao, K. In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative stress-inducible regulon in Escherichia coli. FEBS Lett. 457, 90–92 (1999).

    Article  CAS  Google Scholar 

  11. Choi, H. et al. Structural basis of the redox switch in the OxyR transcription factor. Cell 105, 103–113 (2001).

    Article  CAS  Google Scholar 

  12. Kim, S.O. et al. OxyR: a molecular code for redox-related signaling. Cell 109, 383–396 (2002).

    Article  CAS  Google Scholar 

  13. Zaim, J. & Kierzek, A.M. The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer. Nucleic Acids Res. 31, 1444–1454 (2003).

    Article  CAS  Google Scholar 

  14. Ellis, H.R. & Poole, L.B. Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 36, 15013–15018 (1997).

    Article  CAS  Google Scholar 

  15. Claiborne, A. et al. Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation. Biochemistry 38, 15407–15416 (1999).

    Article  CAS  Google Scholar 

  16. Chen, Y., & Barkley, M.D. Toward understanding tryptophan fluorescence in proteins. Biochemistry 37, 9976–9982 (1998).

    Article  CAS  Google Scholar 

  17. Li, S., Rosen, B.P., Borges-Walmsley, M.I. & Walmsley, A.R. Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator. J. Biol. Chem. 277, 25992–26002 (2003).

    Article  Google Scholar 

  18. Kafri, G. & Horovitz, A. Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1. J. Mol. Biol. 326, 981–987 (2003).

    Article  CAS  Google Scholar 

  19. Grantcharova, V., Alm E.J., Baker, D. & Horwich, A.L. Mechanisms of protein folding. Curr. Opin. Struct. Biol. 11, 70–82 (2001).

    Article  CAS  Google Scholar 

  20. Georgiou, G. How to flip the (redox) switch. Cell 111, 607–610 (2002).

    Article  CAS  Google Scholar 

  21. Ptitsyn, O.B. Molten globule and protein folding. Adv. Protein Chem. 47, 83–229 (1995).

    Article  CAS  Google Scholar 

  22. Santra, M.K. and Panda, D. Detection of an intermediate during unfolding of bacterial cell division protein FtsZ. J. Biol. Chem. 278, 21336–21343 (2003).

    Article  CAS  Google Scholar 

  23. Carr, C.M. & Kim, P.S. A spring-loaded mechanism for the conformational change of influenza hemagglutinin. Cell 73, 823–832 (1993).

    Article  CAS  Google Scholar 

  24. Lee, C., Park, S.H., Lee, M.Y. & Yu, M.H. Regulation of protein function by native metastability. Proc. Natl. Acad. Sci. USA 97, 7727–7731 (2000).

    Article  CAS  Google Scholar 

  25. Fersht, A. In Structure and Mechanism in Protein Science (Freeman, New York, 1998).

    Google Scholar 

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Acknowledgements

We thank A. Matouschek for helpful suggestions on the equilibrium unfolding study, and W. Outten for comments on the manuscript. This research was supported by the National Creative Research Initiative Program (MOST, Korea) and the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program.

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Authors and Affiliations

  1. Center for Cellular Switch Protein Structure, Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yuseong-gu, Daejeon, 305-806, Korea

    Cheolju Lee, Soon Mi Lee, Seung Jun Kim & Seong Eon Ryu

  2. Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Euh-eun-dong, Yuseong-gu, Daejeon, 305-806, Korea

    Seung Jun Kim, Sang Chul Lee & Seong Eon Ryu

  3. Life Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, 130-650, Korea

    Cheolju Lee & Woo-Sung Ahn

  4. Functional Proteomics Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul, 130-650, Korea

    Myeong-Hee Yu

  5. Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Partha Mukhopadhyay & Gisela Storz

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  1. Cheolju Lee
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Supplementary information

Supplementary Table 1

Sensitivities of OxyR C199S and C208S single mutant strains and C199S C208S double mutant strain to H2O2. (PDF 51 kb)

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Lee, C., Lee, S., Mukhopadhyay, P. et al. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat Struct Mol Biol 11, 1179–1185 (2004). https://doi.org/10.1038/nsmb856

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