JBIC Journal of Biological Inorganic Chemistry

, Volume 12, Issue 2, pp 234–247 | Cite as

Phosphorylation-dependent metal binding by α-synuclein peptide fragments

  • Lucy L. Liu
  • Katherine J. Franz
Original Paper


α-Synuclein (α-syn) is the major protein component of the insoluble fibrils that make up Lewy bodies, the hallmark lesions of Parkinson’s disease. Its C-terminal region contains motifs of charged amino acids that potentially bind metal ions, as well as several identified phosphorylation sites. We have investigated the metal-binding properties of synthetic model peptides and phosphopeptides that correspond to residues 119–132 of the C-terminal, polyacidic stretch of human α-syn, with the sequence Ac-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Met-Pro-Ser-Glu-Glu-Gly (α-syn119–132). The peptide pY125 replaces tyrosine with phosphotyrosine, whereas pS129 replaces serine with phosphoserine. By using Tb3+ as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb3+ compared with pS129 and α-syn119–132, a result confirmed by isothermal titration calorimetry. Truncated or alanine-substituted peptides show that the phosphoester group on tyrosine provides a metal-binding anchor that is supplemented by carboxylic acid groups at positions 119, 121, and 126 to establish a multidentate ligand, while two glutamic acid residues at positions 130 and 131 contribute to binding additional Tb3+ ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry, which confirmed that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe3+ and Al3+ induce peptide dimerization through metal ion cross-links. Circular dichroism showed that Fe3+ can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog. The results of this study show that the type and location of a phosphorylated amino acid influence a peptide’s metal-binding specificity and affinity as well as its overall conformation.


Peptide Binding affinity Mass spectrometry Luminescence Protein engineering 



We are grateful for support provided by a National Science Foundation CAREER award (CHE-0449699). We thank Eric J. Toone, Andrea Luteran, and Trine Christensen for help with ITC experiments, and David A. Franz of Lycoming College for many helpful discussions.

Supplementary material


  1. 1.
    Connor JR (ed) (1997) Metals and oxidative damage in neurological disorders. Plenum, New YorkGoogle Scholar
  2. 2.
    Zecca L, Youdim MBH, Riederer P, Connor JR, Crichton RR (2004) Nat Rev Neurosci 5:863–873PubMedCrossRefGoogle Scholar
  3. 3.
    Barnham KJ, Masters CL, Bush AI (2004) Nat Rev Drug Discov 3:205–214PubMedCrossRefGoogle Scholar
  4. 4.
    Brown RC, Lockwood AH, Sonawane BR (2005) Environ Health Perspect 113:1250–1256PubMedCrossRefGoogle Scholar
  5. 5.
    Castellani RJ, Siedlak SL, Perry G, Smith MA (2000) Acta Neuropathol 100:111–114PubMedCrossRefGoogle Scholar
  6. 6.
    Hirsch EC, Brandel JP, Galle P, Javoyagid F, Agid Y (1991) J Neurochem 56:446–451PubMedCrossRefGoogle Scholar
  7. 7.
    Kahle PJ, Haass C, Kretzschmar HA, Neumann M (2002) J Neurochem 82:449–457PubMedCrossRefGoogle Scholar
  8. 8.
    Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L (2000) Science 287:1265–1269PubMedCrossRefGoogle Scholar
  9. 9.
    Giasson BI, Duda JE, Quinn SM, Zhang B, Trojanowski JQ, Lee VM (2002) Neuron 34:521–533PubMedCrossRefGoogle Scholar
  10. 10.
    Feany MB, Bender WW (2000) Nature 404:394–398PubMedCrossRefGoogle Scholar
  11. 11.
    Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PTJ (1996) Biochemistry 35:13709–13715PubMedCrossRefGoogle Scholar
  12. 12.
    Clayton DF, George JM (1999) J Neurosci Res 58:120–129PubMedCrossRefGoogle Scholar
  13. 13.
    Uversky VN, Li J, Fink AL (2001) J Biol Chem 276:44284–44296PubMedCrossRefGoogle Scholar
  14. 14.
    Yamin G, Glaser CB, Uversky VN, Fink AL (2003) J Biol Chem 278:27630–27635PubMedCrossRefGoogle Scholar
  15. 15.
    Paik SR, Shin H.-J, Lee J.-H, Chang C.-S, Kim J (1999) Biochem J 340:821–828PubMedCrossRefGoogle Scholar
  16. 16.
    Nielsen MS, Vorum H, Lindersson E, Jensen PH (2001) J Biol Chem 276:22680–22684PubMedCrossRefGoogle Scholar
  17. 17.
    Lowe R, Pountney DL, Jensen PH, Gai WP, Voelcker NH (2004) Protein Sci 13:3245–3252PubMedCrossRefGoogle Scholar
  18. 18.
    Rasia RM, Bertoncini CW, Marsh D, Hoyer W, Cherny D, Zweckstetter M, Griesinger C, Jovin TM, Fernández CO (2005) Proc Natl Acad Sci USA 102:4294–4299PubMedCrossRefGoogle Scholar
  19. 19.
    Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, Goldberg MS, Shen J, Takio K, Iwatsubo T (2002) Nat Cell Biol 4:160–164PubMedCrossRefGoogle Scholar
  20. 20.
    Ellis CE, Schwartzberg PL, Grider TL, Fink DW, Nussbaum RL (2001) J Biol Chem 276:3879–3884PubMedCrossRefGoogle Scholar
  21. 21.
    Okochi M, Walter J, Koyama A, Nakajo S, Baba M, Iwatsubo T, Meijer L, Kahle PJ Haass C (2000) J Biol Chem 275:390–397PubMedCrossRefGoogle Scholar
  22. 22.
    Pronin AN, Morris AJ, Surguchov A, Benovic JL (2000) J Biol Chem 275:26515–26522PubMedCrossRefGoogle Scholar
  23. 23.
    Takahashi M, Kanuka H, Fujiwara H, Koyama A, Hasegawa M, Miura M, Iwatsubo T (2003) Neurosci Lett 336:155–158PubMedCrossRefGoogle Scholar
  24. 24.
    Nakamura T, Yamashita H, Takahashi T, Nakamura S (2001) Biochem Biophys Res Commun 280:1085–1092PubMedCrossRefGoogle Scholar
  25. 25.
    Mirzaei H, Schieler JL, Rochet JC, Regnier F (2006) Anal Chem 78:2422–2431PubMedCrossRefGoogle Scholar
  26. 26.
    Tholey A, Lindemann A, Kinzel V, Reed J (1999) Biophys J 76:76–87PubMedGoogle Scholar
  27. 27.
    Andrew CD, Warwicker J, Jones GR, Doig AJ (2002) Biochemistry 41:1897–1905PubMedCrossRefGoogle Scholar
  28. 28.
    Errington N, Doig AJ (2005) Biochemistry 44:7553–7558PubMedCrossRefGoogle Scholar
  29. 29.
    Bielska AA, Zondlo NJ (2006) Biochemistry 45:5527–5537PubMedCrossRefGoogle Scholar
  30. 30.
    Signarvic RS, DeGrado WF (2003) J Mol Biol 334:1–12PubMedCrossRefGoogle Scholar
  31. 31.
    Hegenauer J, Saltman P, Nace G (1979) Biochemistry 18:3865–3879PubMedCrossRefGoogle Scholar
  32. 32.
    George A, Bannon L, Sabsay B, Dillon JW, Malone J, Veis A, Jenkins NA, Gilbert DJ, Copel NG (1996) J Biol Chem 271:32869–32873PubMedCrossRefGoogle Scholar
  33. 33.
    Gericke A, Qin C, Spevak L, Fujimoto Y, Butler WT, Sorensen ES, Boskey AL (2005) Calcif Tissue Int 77:45–54PubMedCrossRefGoogle Scholar
  34. 34.
    Yamamoto A, Shin R.-W, Hasegawa K, Naiki H, Sato H, Yoshimasu F, Kitamoto T (2002) J Neurochem 82:1137–1147PubMedCrossRefGoogle Scholar
  35. 35.
    Hollósi M, Urge L, Perczel A, Kajtár J, Teplán I, Otvös L, Fasman GD (1992) J Mol Biol 223:673–682PubMedCrossRefGoogle Scholar
  36. 36.
    Shen ZM, Perczel A, Hollósi M, Nagypál I, Fasman GD (1994) Biochemistry 33:9627–9636PubMedCrossRefGoogle Scholar
  37. 37.
    Liu LL, Franz KJ (2005) J Am Chem Soc 127:9662–9663PubMedCrossRefGoogle Scholar
  38. 38.
    Balakrishnan S, Zondlo NJ (2006) J Am Chem Soc 128:5590–5591PubMedCrossRefGoogle Scholar
  39. 39.
    Cooper JA, Sefton BM, Hunter T (1983) Methods Enzymol 99:387–403PubMedCrossRefGoogle Scholar
  40. 40.
    Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) Protein Sci 4:2411–2423PubMedCrossRefGoogle Scholar
  41. 41.
    Pribil R (1967) Talanta 14:619–627CrossRefPubMedGoogle Scholar
  42. 42.
    Gampp H, Maeder M, Meyer CJ, Zuberbüehler AD (1985) Talanta 32:257–264Google Scholar
  43. 43.
    Vàzquez-Ibar JL, Weinglass AB, Kaback HR (2002) Proc Natl Acad Sci USA 99:3487–3492PubMedCrossRefGoogle Scholar
  44. 44.
    Nakamura S, Yamashita H, Nagano Y, Takahashi T, Avraham S, Avraham H, Matsumoto M, Nakamura S (2002) FEBS Lett 521:190–194PubMedCrossRefGoogle Scholar
  45. 45.
    Takahashi T, Yamashita H, Nagano Y, Nakamura T, Ohmori H, Avraham H, Avraham S, Yasuda M, Matsumoto M (2003) J Biol Chem 278:42225–42233PubMedCrossRefGoogle Scholar
  46. 46.
    Negro A, Brunati AM, Donella-Deana A, Massimino ML, Pinna LA (2001) FASEB J 16:210–212PubMedGoogle Scholar
  47. 47.
    Richardson FS (1982) Chem Rev 82:541–552CrossRefGoogle Scholar
  48. 48.
    Ojida A, Mito-oka Y, Sada K, Hamachi I (2004) J Am Chem Soc 126:2454–2463PubMedCrossRefGoogle Scholar
  49. 49.
    Herrero LA, Terron A (2000) J Biol Inorg Chem 5:269–275PubMedCrossRefGoogle Scholar
  50. 50.
    Hathout Y, Fabris D, Fenselau C (2001) Int J Mass Spectrom 204:1–6CrossRefGoogle Scholar
  51. 51.
    Kramer ML, Kratzin HD, Schmidt B, Römer A, Windl O, Liemann S, Hornemann S, Kretzschmar H (2001) J Biol Chem 276:16711–16719PubMedCrossRefGoogle Scholar
  52. 52.
    Veenstra T, Johnson KL, Tomlinson AJ, Naylor S, Kumar R (1997) Biochemistry 36:3535–3542PubMedCrossRefGoogle Scholar
  53. 53.
    Urvoas A, Amekraz B, Moulin C, Le Clainche L, Stöcklin R, Moutiez M (2003) Rapid Commun Mass Spectrom 17:1889–1896PubMedCrossRefGoogle Scholar
  54. 54.
    Dexter DT, Wells FR, Lees AJ, Agid F, Agid Y, Jenner P, Marsden CD (1989) J Neurochem 52:1830–1836PubMedCrossRefGoogle Scholar
  55. 55.
    Dexter DT, Carayon A, Javoy-Agid F, Agid Y, Wells FR, Daniel SE, Lees AJ, Jenner P, Marsden CD (1991) Brain 114:1953–1975PubMedCrossRefGoogle Scholar
  56. 56.
    Graham JM, Paley MNJ, Grünewald RA, Hoggard N, Griffiths PD (2000) Brain 123:2423–2431PubMedCrossRefGoogle Scholar
  57. 57.
    Sofic E, Paulus W, Jellinger K, Riederer P, Youdim MBH (1991) J Neurochem 56:978–982PubMedCrossRefGoogle Scholar
  58. 58.
    Thong PSP, Watt F, Ponraj D, Leong SK, He Y, Lee TKY (1999) Nucl Instrum Methods Phys Res Sect B 158:349–355CrossRefGoogle Scholar
  59. 59.
    Cole NB, Murphy DD, Lebowitz J, Di Noto L, Levine RL, Nussbaum RL (2005) J Biol Chem 280:9678–9690PubMedCrossRefGoogle Scholar
  60. 60.
    Ostrerova-Golts N, Petrucelli L, Hardy J, Lee JM, Farrer M, Wolozin B (2000) J Neurosci 20:6048–6054PubMedGoogle Scholar
  61. 61.
    Martin FL, Williamson SJM, Paleologou KE, Hewitt R, El-Agnaf OMA, Allsop D (2003) J Neurochem 87:620–630PubMedCrossRefGoogle Scholar
  62. 62.
    Corain B, Bombi GG Tapparo A, Perazzolo M, Zatta P (1996) Coord Chem Rev 149:11–22Google Scholar
  63. 63.
    Exley C (1999) J Inorg Biochem 76:133–140PubMedCrossRefGoogle Scholar
  64. 64.
    Rubini P, Lakatos A, Champmartin D, Kiss T (2002) Coord Chem Rev 228:137–152CrossRefGoogle Scholar
  65. 65.
    Hollender D, Karoly-Lakatos A, Forgo M, Kortvelyesi T, Dombi G, Majer Z, Holloi M, Kiss T, Odani A (2006) J Inorg Biochem 100:351–361PubMedCrossRefGoogle Scholar
  66. 66.
    Kiss E, Lakatos A, Banyai I, Kiss T (1998) J Inorg Biochem 69:145–151CrossRefGoogle Scholar
  67. 67.
    Sung YH, Rospigliosi C, Eliezer D (2006) Biochim Biophys Acta 1764:5–12PubMedGoogle Scholar
  68. 68.
    Crowther RA, Jakes R, Spillantini MG, Goedert M (1998) FEBS Lett 436:309PubMedCrossRefGoogle Scholar
  69. 69.
    Hoyer W, Cherny D, Subramaniam V, Jovin TM (2004) Biochemistry 43:16233–16242PubMedCrossRefGoogle Scholar
  70. 70.
    Murray IVJ, Giasson BI, Quinn SM, Koppaka V, Axelsen PH, Ischiropoulos H, Trojanowski JQ, Lee VMY (2003) Biochemistry 42:8530–8540PubMedCrossRefGoogle Scholar
  71. 71.
    Bertoncini CW, Jung YS, Fernandez CO, Hoyer W, Griesinger C, Jovin TM, Zweckstetter M (2005) Proc Natl Acad Sci USA 102:1430–1435PubMedCrossRefGoogle Scholar
  72. 72.
    Dedmon MM, Lindorff-Larsen K, Christodoulou J, Vendruscolo M, Dobson CM (2005) J Am Chem Soc 127:476–477PubMedCrossRefGoogle Scholar
  73. 73.
    Bernadó P, Bertoncini CW, Griesinger C, Zweckstetter M, Blackledge M (2005) J Am Chem Soc 127:17968–17969PubMedCrossRefGoogle Scholar
  74. 74.
    Khan A, Ashcroft AE, Higenell V, Korchazhkina OV, Exley C (2005) J Inorg Biochem 99:1920–1927PubMedCrossRefGoogle Scholar
  75. 75.
    Ricchelli F, Buggio R, Drago D, Salmona M, Forloni G, Negro A, Tognon G, Zatta P (2006) Biochemistry 45:6724–6732PubMedCrossRefGoogle Scholar
  76. 76.
    Dong J, Shokes JE, Scott RA, Lynn DG (2006) J Am Chem Soc 128:3540–3542PubMedCrossRefGoogle Scholar
  77. 77.
    Shults MD, Imperiali B (2003) J Am Chem Soc 125:14248–14249PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2006

Authors and Affiliations

  1. 1.Department of ChemistryDuke UniversityDurhamUSA

Personalised recommendations