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Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein

Nature Chemical Biology volume 5, pages 53–59 (2009)Cite this article

Abstract

In Bacillus subtilis, PerR is a metal-dependent sensor of hydrogen peroxide. PerR is a dimeric zinc protein with a regulatory site that coordinates either Fe2+ (PerR-Zn-Fe) or Mn2+ (PerR-Zn-Mn). Though most of the peroxide sensors use cysteines to detect H2O2, it has been shown that reaction of PerR-Zn-Fe with H2O2 leads to the oxidation of one histidine residue. Oxidation of PerR leads to the incorporation of one oxygen atom into His37 or His91. This study presents the crystal structure of the oxidized PerR protein (PerR-Zn-ox), which clearly shows a 2-oxo-histidine residue in position 37. Formation of 2-oxo-histidine is demonstrated and quantified by HPLC-MS/MS. EPR experiments indicate that PerR-Zn-H37ox retains a significant affinity for the regulatory metal, whereas PerR-Zn-H91ox shows a considerably reduced affinity for the metal ion. In spite of these major differences in terms of metal binding affinity, oxidation of His37 and/or His91 in PerR prevents DNA binding.

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Figure 1: Crystal structure of the oxidized PerR-Zn-ox protein.
Figure 2: HPLC-MS/MS analysis of the 2-oxo-histidine/histidine ratio for the completely hydrolyzed PerR-Zn-apo and PerR-Zn-ox proteins.
Figure 3: DNA binding affinity of PerR-Zn-apo, PerR-Zn-ox, PerR-Zn-H37A and PerR-Zn-H91A in the presence of Mn2+.
Figure 4: Room temperature EPR analysis of Mn2+ binding by PerR-Zn-ox.
Figure 5: Structural model of PerR regulatory site and oxidation-dependent conformation of PerR.

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Acknowledgements

We thank O. Sénèque for the calibration of the 2-oxo-histidine standard solution by NMR and C. Saint-Pierre for mass spectrometry measurements at the initial stage of this work. This work is dedicated to the memory of Lilian Jacquamet.

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Author notes

  1. Lilian Jacquamet: Deceased.

Authors and Affiliations

  1. Commissariat à l'Energie Atomique, Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Chimie et Biologie des Métaux, CEA-Grenoble, 17 avenue des Martyrs, Grenoble, 38054, Cedex 9, France

    Daouda A K Traoré, Abdelnasser El Ghazouani, Geneviève Blondin, Christelle Caux-Thang, Victor Duarte & Jean-Marc Latour

  2. Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5249, Commissariat à l'Energie Atomique, Université Joseph Fourier, CEA-Grenoble, 17 avenue des Martyrs, Grenoble, 38054, Cedex 9, France

    Daouda A K Traoré, Abdelnasser El Ghazouani, Geneviève Blondin, Christelle Caux-Thang, Victor Duarte & Jean-Marc Latour

  3. Commissariat à l'Energie Atomique, Institut de Biologie Structurale, Laboratoire de Cristallographie et Cristallogenèse des Protéines, Groupe Synchrotron, 17 avenue des Martyrs, Grenoble, 38054, Cedex 9, France

    Daouda A K Traoré, Lilian Jacquamet, Franck Borel & Jean-Luc Ferrer

  4. Unité Mixte de Recherche 5075 Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Joseph Fourier, 41 rue Jules Horowitz, Grenoble, 38027, Cedex 1, France

    Daouda A K Traoré, Lilian Jacquamet, Franck Borel, Jean-Luc Ferrer & David Lascoux

  5. Commissariat à l'Energie Atomique, Institut de Biologie Structurale, Laboratoire de Spectrométrie de Masse des Protéines, 41 rue Jules Horowitz, Grenoble, 38027, Cedex 1, France

    David Lascoux

  6. Commissariat à l'Energie Atomique, Institut Nanosciences et Cryogénie, Service de Chimie Inorganique et Biologique, Lésions des Acides Nucléiques, CEA-Grenoble, 17 avenue des Martyrs, Grenoble, 38054, Cedex 9, France

    Jean-Luc Ravanat

  7. Laboratoire de Chimie Inorganique et Biologique, Unité Mixte de Recherche, E3 Commissariat à l'Energie Atomique, Université Joseph Fourier, 17 avenue des Martyrs, Grenoble, 38054, Cedex 9, France

    Jean-Luc Ravanat

  8. Commissariat à l'Energie Atomique, Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire d'Etude de la Dynamique des Protéines, CEA-Grenoble, 17 avenue des Martyrs, Grenoble, 38054, Cedex 9, France

    Michel Jaquinod

  9. Institut National de la Santé et de la Recherche Médicale U880, Laboratoire d'Etude de la Dynamique des Protéines, CEA-Grenoble, 17 avenue des Martyrs, Grenoble, 38054, Cedex 9, France

    Michel Jaquinod

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  1. Daouda A K Traoré
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Contributions

V.D. and J.-M.L. analyzed the results and co-wrote the paper; C.C.-T. prepared the protein samples; A.E.G. prepared a calibrated solution of 2-oxo-histidine; G.B. performed the EPR experiments, analyzed the results and wrote the corresponding section; D.L., J.-L.R. and M.J. performed the mass spectrometry experiments and analyzed the corresponding results; L.J., D.A.K.T., F.B. and J.-L.F. solved the PerR-Zn-ox structure and wrote the corresponding section.

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Correspondence to Victor Duarte or Jean-Marc Latour.

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods (PDF 686 kb)

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Traoré, D., Ghazouani, A., Jacquamet, L. et al. Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein. Nat Chem Biol 5, 53–59 (2009). https://doi.org/10.1038/nchembio.133

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