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Microbial metalloproteomes are largely uncharacterized

Nature volume 466, pages 779–782 (2010)Cite this article

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Abstract

Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability1,2. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized2,3,4. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

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Figure 1: Metal assimilation by P. furiosus and unassigned metal peaks.
Figure 2: Metal concentration profiles after chromatographic fractionation of P. furiosus cytoplasmic extract.

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References

  1. Gray, H. B., Stiefel, E. I., Valentine, J. S. & Bertini, I. Biological Inorganic Chemistry: Structure and Reactivity (Univ. Science Books, 2006)

    Google Scholar 

  2. Messerschmidt, A., Huber, R., Wieghart, K. & Poulos, T. Handbook of Metalloproteins, Vol. 1–3. (Wiley, 2005)

    Google Scholar 

  3. Shu, N., Zhou, T. & Hovmoller, S. Prediction of zinc-binding sites in proteins from sequence. Bioinformatics 24, 775–782 (2008)

    Article  CAS  Google Scholar 

  4. Kasampalidis, I. N., Pitas, I. & Lyroudia, K. Conservation of metal-coordinating residues. Proteins: Struct. Funct. Bioinf. 68, 123–130 (2007)

    Article  CAS  Google Scholar 

  5. Castagnetto, J. M. et al. MDB: the metalloprotein database and browser at the Scripps Research Institute. Nucleic Acids Res. 30, 379–382 (2002)

    Article  CAS  Google Scholar 

  6. Fan, L. et al. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133, 789–800 (2008)

    Article  CAS  Google Scholar 

  7. Andreini, C., Bertini, I., Cavallaro, G., Holliday, G. L. & Thornton, J. M. Metal-MACiE: a database of metals involved in biological catalysis. Bioinformatics 25, 2088–2089 (2009)

    Article  CAS  Google Scholar 

  8. Waldron, K. J., Rutherford, J. C., Ford, D. & Robinson, N. J. Metalloproteins and metal sensing. Nature 460, 823–830 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Zhang, Y. & Gladyshev, V. N. General trends in trace element utilization revealed by comparative genomic analyses of Co, Cu, Mo, Ni, and Se. J. Biol. Chem. 285, 3393–3405 (2010)

    Article  CAS  Google Scholar 

  10. Lobinski, R., Schaumlöffel, D. & Szpunar, J. Mass spectrometry in bioinorganic analytical chemistry. Mass Spec. Rev. 25, 255–289 (2006)

    Article  ADS  CAS  Google Scholar 

  11. Sanz-Medel, A., Montes-Bayón, M., del Rosario Fernández de la Campa, M., Encinar, J. R. & Bettmer, J. Elemental mass spectrometry for quantitative proteomics. Analyt. Bioanalyt. Chem. 390, 3–16 (2008)

    Article  CAS  Google Scholar 

  12. Shi, W. et al. Metalloproteomics: high-throughput structural and functional annotation of proteins in structural genomics. Structure 13, 1473–1486 (2005)

    Article  CAS  Google Scholar 

  13. Atanassova, A., Högbom, M. & Zamble, D. B. in Methods in molecular biology Vol. 436 (eds B. Kobe, M. Guss & T. Huber) 319–330 (Humana Press, 2008)

    Google Scholar 

  14. Jenney, F. E. & Adams, M. W. W. Rubredoxin from Pyrococcus furiosus . Methods Enzymol. 334, 45–55 (2001)

    Article  CAS  Google Scholar 

  15. Chai, S. C., Wang, W. L. & Ye, Q. Z. Fe(II) is the native cofactor for Escherichia coli methionine aminopeptidase. J. Biol. Chem. 283, 26879–26885 (2008)

    Article  CAS  Google Scholar 

  16. Fiala, G. & Stetter, K. O. Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C. Arch. Microbiol. 145, 56–61 (1986)

    Article  CAS  Google Scholar 

  17. Aiuppa, A., Dongarra, G., Capasso, G. & Allard, P. Trace elements in the thermal groundwaters of Vulcano island (Sicily). J. Volc. Geoth. Res. 98, 189–207 (2000)

    Article  ADS  CAS  Google Scholar 

  18. Menon, A. L. et al. Novel protein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome. Mol. Cell. Proteomics 8, 735–751 (2009)

    Article  CAS  Google Scholar 

  19. Poole, F. L., II et al. Defining genes in the genome of the hyperthermophilic archaeon Pyrococcus furiosus: implications for all microbial genomes. J. Bacteriol. 187, 7325–7332 (2005)

    Article  CAS  Google Scholar 

  20. Hunter, S. et al. InterPro: the integrative protein signature database. Nucleic Acids Res. 37, D211–D215 (2009)

    Article  CAS  Google Scholar 

  21. Agarwal, G., Rajavel, M., Gopal, B. & Srinivasan, N. Structure-based phylogeny as a diagnostic for functional characterization of proteins with a cupin fold. PLoS ONE 4, e5736 (2009)

    Article  ADS  Google Scholar 

  22. Luttringer, F., Mulliez, E., Dublet, B., Lemaire, D. & Fontecave, M. The Zn center of the anaerobic ribonucleotide reductase from E. coli . J. Biol. Inorg. Chem. 14, 923–933 (2009)

    Article  CAS  Google Scholar 

  23. Weinberg, M. V., Schut, G. J., Brehm, S., Datta, S. & Adams, M. W. W. Cold shock of a hyperthermophilic archaeon: Pyrococcus furiosus exhibits multiple responses to a suboptimal growth temperature with a key role for membrane-bound glycoproteins. J. Bacteriol. 187, 336–348 (2005)

    Article  CAS  Google Scholar 

  24. Schwarz, G., Mendel, R. R. & Ribbe, M. W. Molybdenum cofactors, enzymes and pathways. Nature 460, 839–847 (2009)

    Article  ADS  CAS  Google Scholar 

  25. Splan, K. E., Musier-Forsyth, K., Boniecki, M. T. & Martinis, S. A. In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity. Methods 44, 119–128 (2008)

    Article  CAS  Google Scholar 

  26. Ragsdale, S. W. Nickel-based enzyme systems. J. Biol. Chem. 284, 18571–18575 (2009)

    Article  CAS  Google Scholar 

  27. Perry, J. J., Shin, D. S., Getzoff, E. D. & Tainer, J. A. The structural biochemistry of the superoxide dismutases. Biochim. Biophys. Acta 1804, 245–262 (2010)

    Article  CAS  Google Scholar 

  28. Zillig, W. et al. The Sulfolobus-“Caldariella” group: taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch. Microbiol. 125, 259–269 (1980)

    Article  CAS  Google Scholar 

  29. Kosnett, M. J. in Basic and clinical pharmacology 10th ed. (ed. B. G. Katzung) 945–957 (McGraw-Hill, 2007)

    Google Scholar 

  30. Bressler, J. P. et al. Metal transporters in intestine and brain: their involvement in metal-associated neurotoxicities. Hum. Exp. Toxicol. 26, 221–229 (2007)

    Article  CAS  Google Scholar 

  31. Adams, M. W. W. et al. Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus . J. Bacteriol. 183, 716–724 (2001)

    Article  CAS  Google Scholar 

  32. Schut, G. J., Bridger, S. L. & Adams, M. W. W. Insights into the metabolism of elemental sulfur by the hyperthermophilic archaeon Pyrococcus furiosus: characterization of a coenzyme A-dependent NAD(P)H sulfur oxidoreductase. J. Bacteriol. 189, 4431–4441 (2007)

    Article  CAS  Google Scholar 

  33. Cai, Y., Georgiadis, M. & Fourqurean, J. W. Determination of arsenic in seagrass using inductively coupled plasma mass spectrometry. Spectrochim. Acta B 55, 1411–1422 (2000)

    Article  ADS  Google Scholar 

  34. Karthikeyan, S., Joshi, U. M. & Balasubramanian, R. Microwave assisted sample preparation for determining water-soluble fraction of trace elements in urban airborne particulate matter: evaluation of bioavailability. Anal. Chim. Acta 576, 23–30 (2006)

    Article  CAS  Google Scholar 

  35. Blake, P. R. et al. Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR. Biochemistry 30, 10885–10895 (1991)

    Article  CAS  Google Scholar 

  36. Quevillon, E. et al. InterProScan: protein domains identifier. Nucleic Acids Res. 33, W116–W120 (2005)

    Article  CAS  Google Scholar 

  37. Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular cloning: A Laboratory Manual 2nd ed. Vol. 3 (Cold Spring Harbor Laboratory Press, 1989)

    Google Scholar 

Download references

Acknowledgements

This research is part of the MAGGIE (Molecular Assemblies, Genes and Genomes Integrated Efficiently) project supported by Department of Energy grant (DE-FG0207ER64326). We thank S. Hammond, L. Wells, R. Hopkins and D. Phillips for help with in-gel MS analyses.

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Author notes

  1. Francis E. Jenney Jr

    Present address: Present address: Philadelphia College of Osteopathic Medicine, Suwanee, Georgia 30024, USA (F.E.J.).,

  2. Aleksandar Cvetkovic, Angeli Lal Menon and Michael P. Thorgersen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, 30602, Georgia, USA

    Aleksandar Cvetkovic, Angeli Lal Menon, Michael P. Thorgersen, Joseph W. Scott, Farris L. Poole II, Francis E. Jenney Jr, W. Andrew Lancaster, Jeremy L. Praissman, Saratchandra Shanmukh, Brian J. Vaccaro & Michael W. W. Adams

  2. Scripps Center for Mass Spectrometry and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, 92037, California, USA

    Sunia A. Trauger, Ewa Kalisiak, Junefredo V. Apon & Gary Siuzdak

  3. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Steven M. Yannone & John A. Tainer

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Contributions

A.C., A.L.M., M.P.T. and J.W.S. grew and fractionated P. furiosus; A.L.M. carried out cytoplasmic washes; A.L.M. and S.M.Y. grew and fractionated S. solfataricus; A.L.M. and M.P.T. grew and fractionated E. coli; A.C. and S.S. performed ICP-MS analyses; S.A.T., E.K., J.V.A. and G.S. performed HT-MS/MS analyses; A.L.M. purified PF0056; J.W.S. purified PF1972 and PF0086; M.P.T. and B.J.V. purified PF0742; M.T.P. purified PF1587, PF0215, PF1343 and PF0257; W.A.L., J.L.P. and F.L.P. carried out metal-protein bioinformatic analyses; A.C., A.L.M., F.E.J., F.L.P., M.P.T. and J.A.T. and M.W.W.A. contributed to experimental design and data analyses, and wrote the paper.

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Correspondence to Michael W. W. Adams.

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Cvetkovic, A., Menon, A., Thorgersen, M. et al. Microbial metalloproteomes are largely uncharacterized. Nature 466, 779–782 (2010). https://doi.org/10.1038/nature09265

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