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Protein and metal cluster structure of the wheat metallothionein domain γ-Ec-1: the second part of the puzzle

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Abstract

Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including ZnII, CdII, and CuI. MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT Ec-1 from Triticum aestivum, common bread wheat, is a ZnII-binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding βE domain was recently described. Here we present the structure of the N-terminal second domain, γ-Ec-1, determined by NMR spectroscopy. The γ-Ec-1 domain enfolds an M II2 Cys6 cluster and was characterized as part of the full-length Zn6Ec-1 protein as well as in the form of the separately expressed domain, both in the ZnII-containing isoform and the CdII-containing isoform. Extended X-ray absorption fine structure analysis of Zn2γ-Ec-1 clearly shows the presence of a ZnS4 coordination sphere with average Zn–S distances of 2.33 Å. 113Cd NMR experiments were used to identify the MII-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of γ-Ec-1 were probed with CdII binding experiments as well as by pH titrations of the ZnII and CdII forms, the latter suggesting an interaction of the γ domain and the βE domain within the full-length protein.

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Acknowledgments

We thank Peter Güntert for refining the CYANA structures with a full force field. This work was supported by the Swiss National Science Foundation (SNSF Professorship PP002-119106/1 to E.F.).

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Authors and Affiliations

  1. Institute of Inorganic Chemistry, University of Zurich, 8057, Zurich, Switzerland

    Jens Loebus, Estevão A. Peroza, Nancy Blüthgen, Thomas Fox & Eva Freisinger

  2. European Molecular Biology Laboratory (EMBL), Outstation Hamburg at Deutsches Elektronen-Synchrotron (DESY), 22603, Hamburg, Germany

    Wolfram Meyer-Klaucke

  3. Institute of Organic Chemistry, University of Zurich, 8057, Zurich, Switzerland

    Oliver Zerbe

Authors
  1. Jens Loebus
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  2. Estevão A. Peroza
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  3. Nancy Blüthgen
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  4. Thomas Fox
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  5. Wolfram Meyer-Klaucke
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  6. Oliver Zerbe
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  7. Eva Freisinger
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Corresponding authors

Correspondence to Oliver Zerbe or Eva Freisinger.

Additional information

J. Loebus and E. A. Peroza contributed equally.

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Loebus, J., Peroza, E.A., Blüthgen, N. et al. Protein and metal cluster structure of the wheat metallothionein domain γ-Ec-1: the second part of the puzzle. J Biol Inorg Chem 16, 683–694 (2011). https://doi.org/10.1007/s00775-011-0770-2

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