Protein-folding location can regulate manganese-binding versus copper- or zinc-binding

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Metals are needed by at least one-quarter of all proteins1,2. Although metallochaperones3,4,5,6,7,8 insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving–Williams series9, Cu2+ and Zn2+ typically form more stable complexes than Mn2+. Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu2+-protein, CucA (Cu2+-cupin A), and the most abundant Mn2+-protein, MncA (Mn2+-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving–Williams series, MncA only binds Mn2+ after folding in solutions containing at least a 104 times molar excess of Mn2+ over Cu2+ or Zn2+. However once MncA has bound Mn2+, the metal does not exchange with Cu2+. MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn2+ (refs 10–12) but micromolar Mn2+ (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu2+. These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn2+.

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Figure 1: CucA is the principal periplasmic Cu 2+ -protein.
Figure 2: MncA is the principal periplasmic Mn 2+ -protein.
Figure 3: MncA and CucA have similar metal-sites.
Figure 4: MncA prefers Cu2+ but entraps Mn2+.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The crystallographic coordinates of MncA have been deposited in the Protein Data Bank under accession number 2VQA.


  1. 1

    Rosenzweig, A. C. Metallochaperones: Bind and deliver. Chem. Biol. 9, 673–677 (2002)

  2. 2

    Ferrer, M., Golyshina, O. V., Beloqui, A., Golyshin, P. N. & Timmis, K. N. The cellular machinery of Ferroplasma acidiphilum is iron-protein-dominated. Nature 445, 91–94 (2007)

  3. 3

    Odermatt, A. & Solioz, M. Two trans-acting metalloregulatory proteins controlling expression of the copper-ATPases of Enterococcus hirae . J. Biol. Chem. 270, 4349–4354 (1995)

  4. 4

    Beers, J., Glerum, D. M. & Tzagoloff, A. Purification, characterisation, and localisation of yeast Cox17p, a mitochondrial copper shuttle. J. Biol. Chem. 272, 33191–33196 (1997)

  5. 5

    Carr, S. & Winge, D. R. Assembly of cytochrome c oxidase within the mitochondrion. Acc. Chem. Res. 36, 309–316 (2003)

  6. 6

    Pufahl, R. A. et al. Metal ion chaperone function of the soluble CuI receptor Atx1. Science 278, 853–856 (1997)

  7. 7

    Valentine, J. S. & Gralla, E. B. Delivering copper inside yeast and human cells. Science 278, 817–818 (1997)

  8. 8

    Banci, L. et al. The Atx1-Ccc2 complex is a metal-mediated protein-protein interaction. Nature Chem. Biol. 2, 367–368 (2006)

  9. 9

    Fraústo da Silva, J. J. R. & Williams, R. J. P. The Biological Chemistry of the Elements: The Inorganic Chemistry of Life 2nd edn (Clarendon, 2001)

  10. 10

    Changela, A. et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383–1387 (2003)

  11. 11

    Rae, T. D., Schmidt, P. J., Pufahl, R. A., Culotta, V. C. & O'Halloran, T. V. Undetectable intracellular free copper: The requirement of a copper chaperone for superoxide dismutase. Science 284, 805–808 (1999)

  12. 12

    Outten, C. E. & O'Halloran, T. V. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 292, 2488–2492 (2001)

  13. 13

    Helmann, J. D. Measuring metals with RNA. Mol. Cell 27, 859–860 (2007)

  14. 14

    Tottey, S., Harvie, D. R. & Robinson, N. J. in Molecular Microbiology of Heavy Metals (eds Nies, D. H. & Silver, S.) 1–35 (Microbiology Monographs Vol. 6, Springer, 2007)

  15. 15

    Fusetti, F. et al. Crystal structure of the copper-containing quercetin dioxygenase from Aspergillus japonicus . Structure 10, 259–268 (2002)

  16. 16

    Tanner, A., Bowater, L., Fairhurst, S. A. & Bornemann, S. Oxalate decarboxylase requires manganese and dioxygen for activity. J. Biol. Chem. 276, 43627–43634 (2001)

  17. 17

    Dunwell, J. M., Khuri, S. & Gane, P. J. Microbial relatives of the seed storage proteins of higher plants: Conservation of structure and diversification of function during evolution of the cupin superfamily. Microbiol. Mol. Biol. Rev. 64, 153–179 (2000)

  18. 18

    Kooter, I. M. et al. EPR characterization of the mononuclear Cu-containing Aspergillus japonicus quercetin 2,3-dioxygenase reveals dramatic changes upon anaerobic binding of substrates. Eur. J. Biochem. 269, 2971–2979 (2002)

  19. 19

    Angerhofer, A. et al. Multifrequency EPR studies on the Mn(II) centers of oxalate decarboxylase. J. Phys. Chem. B 111, 5043–5046 (2007)

  20. 20

    Fulda, S., Huang, F., Nilsson, F., Hagemann, M. & Norling, B. Proteomics of Synechocystis PCC 6803: Identification of periplasm proteins in cells grown at low and high salt concentrations. Eur. J. Biochem. 267, 5900–5907 (2000)

  21. 21

    Robinson, C. & Bolhuis, A. Tat-dependent protein targeting in prokaryotes and chloroplasts. Biochim. Biophys. Acta 1694, 135–147 (2004)

  22. 22

    Tottey, S. et al. A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition. J. Biol. Chem. 277, 5490–5497 (2002)

  23. 23

    Santini, C. L. et al. A novel sec-independent periplasmic protein translocation pathway in Escherichia coli . EMBO J. 17, 101–112 (1998)

  24. 24

    Palmer, T. & Berks, B. C. Moving folded proteins across the bacterial cell membrane. Microbiology 149, 547–556 (2003)

  25. 25

    Keegstra, K. & Cline, K. Protein import and routing systems of chloroplasts. Plant Cell 11, 557–570 (1999)

  26. 26

    Ranquet, C., Ollagnier-de-Choudens, S., Loiseau, L., Barras, L. & Fontecave, M. Cobalt stress in E. coli: The effect on the iron-sulfur proteins. J. Biol. Chem. 282, 30442–30451 (2007)

  27. 27

    Brown, D. R. et al. The cellular prion protein binds copper in vivo . Nature 390, 684–687 (1997)

  28. 28

    Bellingham, S. A. et al. Copper depletion down-regulates expression of the Alzheimer's disease amyloid-beta precursor protein gene. J. Biol. Chem. 279, 20378–20386 (2004)

  29. 29

    Bush, A. I. The metallobiology of Alzheimer's disease. Trends Neurosci. 26, 207–214 (2003)

  30. 30

    Waldron, K. J., Tottey, S., Yanagisawa, S., Dennison, C. & Robinson, N. J. A periplasmic ironbinding protein contributes toward inward copper supply. J. Biol. Chem. 282, 3837–3846 (2007)

  31. 31

    Wold, S., Esbensen, K. & Geladi, P. Principal component analysis. Chemom. Intell. Lab. Syst. 2, 37–52 (1987)

  32. 32

    Orriss, G. L. et al. TatBC, TatB and TatC form structurally autonomous units within the twin arginine transport system of Escherichia coli . FEBS Lett. 581, 4091–4097 (2007)

  33. 33

    DeLisa, M. P., Samuelson, P., Palmer, T. & Georgiou, G. Genetic analysis of the twin arginine translocator secretion pathway in bacteria. J. Biol. Chem. 277, 29825–29831 (2002)

  34. 34

    Leslie, A. G. W. Recent Changes to the MOSFLM Package for Processing Film and Image Plate Data (Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, Number 26, Daresbury Laboratory, Warrington, UK, 1992)

  35. 35

    Kabsch, W. J. Evaluation of single-crystal X-ray diffraction data from a position-sensitive detector. J. Appl. Crystallogr. 21, 916–924 (1988)

  36. 36

    Navaza, J. AMoRe: An automated package for molecular replacement. Acta Crystallogr. A50, 157–163 (1994)

  37. 37

    Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P. & Ealick, S. E. Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 Å resolution. Biochemistry 41, 7659–7669 (2002)

  38. 38

    Schwarzenbacher, R., Godzik, A., Grzechnik, S. K. & Jaroszewski, L. The importance of alignment accuracy for molecular replacement. Acta Crystallogr. D 60, 1229–1236 (2004)

  39. 39

    Emsley, P. & Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. 60, 2126–2132 (2004)

  40. 40

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. 53, 240–255 (1997)

  41. 41

    DeLano, W. The PyMOL Molecular Graphics System (DeLano Scientific, 2002)

  42. 42

    Svedružić, D. et al. Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis . Arch. Biochem. Biophys. 464, 36–47 (2007)

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This work was supported by the BBSRC PMS committee (BBS/B/02576 and BB/E001688/1). B.R. was supported by J. Gitlin and the Children's Discovery Institute. M.J.B. is a Royal Society University Research Fellow. J. Kotz provided advice on the draft manuscript. We thank T. Palmer, B. Ize and G. Buchanan for donating E. coli BL21 tat deletion strains and for advice.

Author Contributions S.T. and K.J.W. contributed equally, doing most of the laboratory work and playing a part in planning and data interpretation. S.J.F. and M.J.B. collected and interpreted the X-ray diffraction data. C.D. and K.S. collected and interpreted the EPR data. B.R. did the metal exchange experiment. J.G. did the mass fingerprinting, C.B. generated the algorithm for principal component analysis and T.R.C. did the confocal microscopy. N.J.R. managed the programme, and had overall responsibility for data interpretation and writing the manuscript.

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Correspondence to Nigel J. Robinson.

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Tottey, S., Waldron, K., Firbank, S. et al. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding. Nature 455, 1138–1142 (2008) doi:10.1038/nature07340

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