Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I)

Abstract

Cellular acquisition of copper in eukaryotic organisms is primarily accomplished through high-affinity copper transport proteins (Ctr). The extracellular N-terminal regions of both human and yeast Ctr1 contain multiple methionine residues organized in copper-binding Mets motifs. These motifs comprise combinations of methionine residues arranged in clusters of MXM and MXXM, where X can be one of several amino acids. Model peptides corresponding to 15 different Mets motifs were synthesized and determined to selectively bind Cu(I) and Ag(I), with no discernible affinity for divalent metal ions. These are rare examples of biological thioether-only metal binding sites. Effective dissociation constant (K D) values for the model Mets peptides and Cu(I) were determined by an ascorbic acid oxidation assay and validated through electrospray ionization mass spectrometry and range between 2 and 11 μM. Affinity appears to be independent of pH, the arrangement of the motif, and the composition of intervening amino acids, all of which reveal the generality and flexibility of the MX1–2MX1–2M domain. Circular dichroism spectroscopy, 1H-NMR spectroscopy, and X-ray absorption spectroscopy were also used to characterize the binding event. These results are intended to aid the development of the still unknown mechanism of copper transport across the cell membrane.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  1. 1.

    Peña MMO, Lee J, Thiele DJ (1999) J Nutr 129:1251–1260

    PubMed  Google Scholar 

  2. 2.

    Macomber L, Imlay JA (2009) Proc Natl Acad Sci USA 106:8344–8349. doi:10.1073/pnas.0812808106

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV (1999) Science 284:805–808

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Bush AI (2003) Trends Neurosci 26:207–214. doi:10.1016/s0166-2236(03)00067-5

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Valentine JS, Hart PJ (2003) Proc Natl Acad Sci USA 100:3617–3622. doi:10.1073/pnas.0730423100

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Puig S, Thiele DJ (2002) Curr Opin Chem Biol 6:171–180

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Puig S, Lee J, Lau M, Thiele DJ (2002) J Biol Chem 277:26021–26030. doi:10.1074/jbc.M202547200

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Xiao Z, Loughlin F, George GN, Howlett GJ, Wedd AG (2004) J Am Chem Soc 126:3081–3090. doi:10.1021/ja0390350

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    De Feo CJ, Aller SG, Siluvai GS, Blackburn NJ, Unger VM (2009) Proc Natl Acad Sci USA 106:4237–4242. doi:10.1073/pnas.0810286106

    Article  PubMed  Google Scholar 

  10. 10.

    Klomp AEM, Juijn JA, Van der Gun LTM, Van den Berg IET, Berger R, Klomp LWJ (2003) Biochem J 370:881–889. doi:10.1042/bj20021128

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Aller SG, Unger VM (2006) Proc Natl Acad Sci USA 103:3627–3632. doi:10.1073/pnas.0509929103

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Cobine PA, Ojeda LD, Rigby KM, Winge DR (2004) J Biol Chem 279:14447–14455. doi:10.1074/jbc.M312693200

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Cobine PA, Pierrel F, Winge DR (2006) Biochim Biophys Acta Mol Cell Res 1763:759–772. doi:10.1016/j.bbamcr.2006.03.002

    CAS  Article  Google Scholar 

  14. 14.

    Nose Y, Rees EM, Thiele DJ (2006) Trends Biochem Sci 31:604–607. doi:10.1016/j.tibs.2006.09.003

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Kim BE, Nevitt T, Thiele DJ (2008) Nat Chem Biol 4:176–185

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Jiang J, Nadas IA, Kim MA, Franz KJ (2005) Inorg Chem 44:9787–9794. doi:10.1021/ic051180m

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Hassett R, Kosman DJ (1995) J Biol Chem 270:128–134

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Dancis A, Yuan DS, Haile D, Askwith C, Eide D, Moehle C, Kaplan J, Klausner RD (1994) Cell 76:393–402

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Georgatsou E, Mavrogiannis LA, Fragiadakis GS, Alexandraki D (1997) J Biol Chem 272:13786–13792

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Rorabacher DB (2004) Chem Rev 104:651–697. doi:10.1021/cr020630e

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Colman PM, Freeman HC, Guss JM, Murata M, Norris VA, Ramshaw JAM, Venkatappa MP (1978) Nature 272:319–324

    CAS  Article  Google Scholar 

  22. 22.

    Norris GE, Anderson BF, Baker EN (1986) J Am Chem Soc 108:2784–2785

    CAS  Article  Google Scholar 

  23. 23.

    Arnesano F, Banci L, Bertini I, Huffman DL, O’Halloran TV (2001) Biochemistry 40:1528–1539

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Banci L, Bertini I, Ciofi-Baffoni S, Huffman DL, O’Halloran TV (2001) J Biol Chem 276:8415–8426

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Schafer FQ, Buettner GR (2001) Free Radic Biol Med 30:1191–1212

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Davis AV, O’Halloran TV (2008) Nat Chem Biol 4:148–151

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Abajian C, Yatsunyk LA, Ramirez BE, Rosenzweig AC (2004) J Biol Chem 279:53584–53592. doi:10.1074/jbc.M408099200

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Elam JS, Thomas ST, Holloway SP, Taylor AB, Hart PJ (2002) Copper-containing proteins. Academic Press, San Diego, pp 151–219

    Google Scholar 

  29. 29.

    George GN, Pickering IJ (2001) EXAFSPAK. http://www-ssrl.slac.stanford.edu/exafspak.html

  30. 30.

    Ankudinov AL, Rehr JJ (2003) J Synchrotron Radiat 10:366–368. doi:10.1107/s0909049503009130

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Lieberman RL, Kondapalli KC, Shrestha DB, Hakemian AS, Smith SM, Telser J, Kuzelka J, Gupta R, Borovik AS, Lippard SH, Hoffman BM, Rosenzweig AC, Stemmler TL (2006) Inorg Chem 45:8372–8381. doi:10.1021/ic060739v

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Wernimont AK, Huffman DL, Finney LA, Demeler B, O’Halloran TV, Rosenzweig AC (2003) J Biol Inorg Chem 8:185–194. doi:10.1007/s00775-002-0404-9

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Labbé S, Peña MMO, Fernandes AR, Thiele DJ (1999) J Biol Chem 274:36252–36260

    Article  PubMed  Google Scholar 

  34. 34.

    Zhou H, Thiele DJ (2001) J Biol Chem 276:20529–20535

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Djoko KY, Xiao ZG, Huffman DL, Wedd AG (2007) Inorg Chem 46:4560–4568. doi:10.1021/ic070107o

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Yatsunyk LA, Rosenzweig AC (2007) J Biol Chem 282:8622–8631. doi:10.1074/jbc.M609533200

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Xiao ZG, Donnelly PS, Zimmermann M, Wedd AG (2008) Inorg Chem 47:4338–4347. doi:10.1021/ic702440e

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Martell AE (1982) Adv Chem Ser 200:153–178

    CAS  Article  Google Scholar 

  39. 39.

    Lim J, Vachet RW (2003) Anal Chem 75:1164–1172. doi:10.1021/ac026206v

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Nadal RC, Abdelraheim SR, Brazier MW, Rigby SEJ, Brown DR, Viles JH (2007) Free Radic Biol Med 42:79–89. doi:10.1016/j.freeradbiomed.2006.09.019

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Nadal RC, Rigby SEJ, Viles JH (2008) Biochemistry 47:11653–11664. doi:10.1021/bi8011093

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Buettner GR (1986) Free Radic Res Commun 1:349–353

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Buettner GR (1988) J Biochem Biophys Methods 16:27–40

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Van der Rest ME, Kamminga AH, Nakano A, Anraku Y, Poolman B, Konings WN (1995) Microbiol Rev 59:304–322

    PubMed  Google Scholar 

  45. 45.

    Lee J, Peña MMO, Nose Y, Thiele DJ (2002) J Biol Chem 277:4380–4387. doi:10.1074/jbc.M104728200

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Khan MMT, Martell AE (1967) J Am Chem Soc 89:7104–7111

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Mi L, Zuberbühler AD (1992) Helv Chim Acta 75:1547–1556

    CAS  Article  Google Scholar 

  48. 48.

    Sisley MJ, Jordan RB (1997) Dalton Trans 3883–3888

  49. 49.

    Perczel A, Hollósi M, Foxman BM, Fasman GD (1991) J Am Chem Soc 113:9772–9784

    CAS  Article  Google Scholar 

  50. 50.

    Arnesano F, Scintilla S, Natile G (2007) Angew Chem Int Ed 46:9062–9064. doi:10.1002/anie.200703271

    CAS  Article  Google Scholar 

  51. 51.

    Kau LS, Spira-Solomon DJ, Penner-Hahn JE, Hodgson KO, Solomon EI (1987) J Am Chem Soc 109:6433–6442

    CAS  Article  Google Scholar 

  52. 52.

    D’Angelo P, Pacello F, Mancini G, Proux O, Hazemann JL, Desideri A, Battistoni A (2005) Biochemistry 44:13144–13150. doi:10.1021/bi050925x

    Article  PubMed  Google Scholar 

  53. 53.

    Bagai I, Liu W, Rensing C, Blackburn NJ, McEvoy MM (2007) J Biol Chem 282:35695–35702. doi:10.1074/jbc.M703937200

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    Sarret G, Favier A, Coves J, Hazemann J-L, Mergeay M, Bersch B (2010) J Am Chem Soc 132:3770–3777

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    Pickering IJ, George GN, Dameron CT, Kurz B, Winge DR, Dance IG (1993) J Am Chem Soc 115:9498–9505

    CAS  Article  Google Scholar 

  56. 56.

    Ralle M, Lutsenko S, Blackburn NJ (2003) J Biol Chem 278:23163–23170. doi:10.1074/jbc.M303474200

    CAS  Article  PubMed  Google Scholar 

  57. 57.

    Chong LX, Ash MR, Maher MJ, Hinds MG, Xiao ZG, Wedd AG (2009) J Am Chem Soc 131:3549–3564. doi:10.1021/ja807354z

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Peariso K, Huffman DL, Penner-Hahn JE, O’Halloran TV (2003) J Am Chem Soc 125:342–343. doi:10.1021/ja028935y

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Kittleson JT, Loftin IR, Hausrath AC, Engelhardt KP, Rensing C, McEvoy MM (2006) Biochemistry 45:11096–11102. doi:10.1021/bi061622

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Loftin IR, Franke S, Blackburn NJ, McEvoy MM (2007) Protein Sci 16:2287–2293. doi:10.1110/ps.073021307

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Xue Y, Davis AV, Balakrishnan G, Stasser JP, Staehlin BM, Focia P, Spiro TG, Penner-Hahn JE, O’Halloran TV (2008) Nat Chem Biol 4:107–109. doi:10.1038/nchembio.2007.57

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Shannon RD (1976) Acta Crystallogr Sect A 32:751–767

    Article  Google Scholar 

  63. 63.

    Dancis A, Haile D, Yuan DS, Klausner RD (1994) J Biol Chem 269:25660–25667

    CAS  PubMed  Google Scholar 

  64. 64.

    Crider SE, Holbrook RJ, Franz KJ (2010) Metallomics 2:74–83. doi:10.1039/b916899k

    CAS  Article  Google Scholar 

  65. 65.

    Ishida S, Lee J, Thiele DJ, Herskowitz I (2002) Proc Natl Acad Sci USA 99:14298–14302. doi:10.1073/pnas.162491399

    CAS  Article  PubMed  Google Scholar 

  66. 66.

    Kim CK, Choi SG, Rho YS (1988) Arch Pharm Res 11:81–86

    CAS  Article  Google Scholar 

  67. 67.

    Gray VA, Dressman JB (1996) Pharmacop Forum 22:1943–1945

    Google Scholar 

  68. 68.

    Zhang L, Koay M, Maher MJ, Xiao Z, Wedd AG (2006) J Am Chem Soc 128:5834–5850. doi:10.1021/ja058528x

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank the National Science Foundation (Grant CAREER 0449699) for funding these studies. K.J.F. also thanks the Sloan Foundation and the Camille and Henry Dreyfus Foundation. We thank Marina Dickens, Anthony Ribeiro, and Ronald Venters for assistance with NMR spectroscopy, and Terry Oas for many helpful discussions. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. P.R.G. was supported by a grant from the Camille and Henry Dreyfus Foundation (Henry Dreyfus Teacher-Scholar Program) and by a grant from the National Institutes of Health to the state of South Carolina as part of NCRR’s INBRE program (P20 RR-016461).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Katherine J. Franz.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rubino, J.T., Riggs-Gelasco, P. & Franz, K.J. Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I). J Biol Inorg Chem 15, 1033–1049 (2010). https://doi.org/10.1007/s00775-010-0663-9

Download citation

Keywords

  • Copper
  • Silver
  • Methionine
  • Copper transport
  • Ctr