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. 2004 May 15;380(Pt 1):273–282. doi: 10.1042/BJ20031767

Copper induces increased beta-sheet content in the scrapie-susceptible ovine prion protein PrPVRQ compared with the resistant allelic variant PrPARR.

Edmond Wong 1, Alana M Thackray 1, Raymond Bujdoso 1
PMCID: PMC1224157  PMID: 14969585

Abstract

Prion diseases are characterized by conformational change in the copper-binding protein PrP (prion protein). Polymorphisms in ovine PrP at amino acid residues 136, 154 and 171 are associated with variation in susceptibility to scrapie. PrPVRQ [PrP(Val136/Arg154/Gln171)] or PrPARQ [PrP(Ala136/Arg154/Gln171)] animals show susceptibility to scrapie, whereas those that express Ala136/Arg154/Arg171 (PrPARR) show resistance. Results are presented here that show PrPVRQ and PrPARR display different conformational responses to metal-ion interaction. At 37 degrees C copper induced different levels of b-sheet content in the allelic variants of ovine full-length prion protein (amino acid 25-232). PrPVRQ showed a significant increase in b-sheet content when exposed to copper at 37 degrees C, whereas PrPARR remained relatively unchanged. The conversion of a-helical PrPVRQ to b-sheet form was shown by CD spectroscopy and the decreased binding of C-terminal specific monoclonal anti-PrP antibodies. This conversion to an increased b-sheet form did not occur with truncated PrPVRQ (amino acids 89-233), which demonstrates that additional metal-binding sites outside of the N-terminus may not overtly influence the overall structure of ovine PrP. Despite the difference in b-sheet content, both the scrapie-susceptible and -resistant allelic forms of ovine PrP acquired resistance to proteinase K digestion following exposure to copper at 37 degrees C, suggesting the potential for disease-associated PrPARR to accumulate in vivo. Our present study demonstrates that allelic variants of ovine PrP differ in their structure and response to the interaction with copper. These observations will contribute to a better understanding of the mechanism of susceptibility and resistance to prion disease.

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Selected References

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  1. Aronoff-Spencer E., Burns C. S., Avdievich N. I., Gerfen G. J., Peisach J., Antholine W. E., Ball H. L., Cohen F. E., Prusiner S. B., Millhauser G. L. Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy. Biochemistry. 2000 Nov 14;39(45):13760–13771. doi: 10.1021/bi001472t. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baskakov I. V., Legname G., Prusiner S. B., Cohen F. E. Folding of prion protein to its native alpha-helical conformation is under kinetic control. J Biol Chem. 2001 Apr 16;276(23):19687–19690. doi: 10.1074/jbc.C100180200. [DOI] [PubMed] [Google Scholar]
  3. Baskakov Ilia V., Legname Giuseppe, Baldwin Michael A., Prusiner Stanley B., Cohen Fred E. Pathway complexity of prion protein assembly into amyloid. J Biol Chem. 2002 Mar 23;277(24):21140–21148. doi: 10.1074/jbc.M111402200. [DOI] [PubMed] [Google Scholar]
  4. Bonomo R. P., Imperllizzeri G., Pappalardo G., Rizzarelli E., Tabbì G. Copper(II) binding modes in the prion octapeptide PHGGGWGQ: a spectroscopic and voltammetric study. Chemistry. 2000 Nov 17;6(22):4195–4202. doi: 10.1002/1521-3765(20001117)6:22<4195::aid-chem4195>3.0.co;2-2. [DOI] [PubMed] [Google Scholar]
  5. Brown D. R., Hafiz F., Glasssmith L. L., Wong B. S., Jones I. M., Clive C., Haswell S. J. Consequences of manganese replacement of copper for prion protein function and proteinase resistance. EMBO J. 2000 Mar 15;19(6):1180–1186. doi: 10.1093/emboj/19.6.1180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown D. R., Qin K., Herms J. W., Madlung A., Manson J., Strome R., Fraser P. E., Kruck T., von Bohlen A., Schulz-Schaeffer W. The cellular prion protein binds copper in vivo. Nature. 1997 Dec 18;390(6661):684–687. doi: 10.1038/37783. [DOI] [PubMed] [Google Scholar]
  7. Brown D. R., Wong B. S., Hafiz F., Clive C., Haswell S. J., Jones I. M. Normal prion protein has an activity like that of superoxide dismutase. Biochem J. 1999 Nov 15;344(Pt 1):1–5. [PMC free article] [PubMed] [Google Scholar]
  8. Burns Colin S., Aronoff-Spencer Eliah, Dunham Christine M., Lario Paula, Avdievich Nikolai I., Antholine William E., Olmstead Marilyn M., Vrielink Alice, Gerfen Gary J., Peisach Jack. Molecular features of the copper binding sites in the octarepeat domain of the prion protein. Biochemistry. 2002 Mar 26;41(12):3991–4001. doi: 10.1021/bi011922x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Buschmann A., Kuczius T., Bodemer W., Groschup M. H. Cellular prion proteins of mammalian species display an intrinsic partial proteinase K resistance. Biochem Biophys Res Commun. 1998 Dec 30;253(3):693–702. doi: 10.1006/bbrc.1998.9838. [DOI] [PubMed] [Google Scholar]
  10. Caughey B., Raymond G. J. The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J Biol Chem. 1991 Sep 25;266(27):18217–18223. [PubMed] [Google Scholar]
  11. Clouscard C., Beaudry P., Elsen J. M., Milan D., Dussaucy M., Bounneau C., Schelcher F., Chatelain J., Launay J. M., Laplanche J. L. Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie. J Gen Virol. 1995 Aug;76(Pt 8):2097–2101. doi: 10.1099/0022-1317-76-8-2097. [DOI] [PubMed] [Google Scholar]
  12. Donne D. G., Viles J. H., Groth D., Mehlhorn I., James T. L., Cohen F. E., Prusiner S. B., Wright P. E., Dyson H. J. Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13452–13457. doi: 10.1073/pnas.94.25.13452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Flechsig E., Shmerling D., Hegyi I., Raeber A. J., Fischer M., Cozzio A., von Mering C., Aguzzi A., Weissmann C. Prion protein devoid of the octapeptide repeat region restores susceptibility to scrapie in PrP knockout mice. Neuron. 2000 Aug;27(2):399–408. doi: 10.1016/s0896-6273(00)00046-5. [DOI] [PubMed] [Google Scholar]
  14. Goldmann W., Hunter N., Smith G., Foster J., Hope J. PrP genotype and agent effects in scrapie: change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie. J Gen Virol. 1994 May;75(Pt 5):989–995. doi: 10.1099/0022-1317-75-5-989. [DOI] [PubMed] [Google Scholar]
  15. Hasnain S. S., Murphy L. M., Strange R. W., Grossmann J. G., Clarke A. R., Jackson G. S., Collinge J. XAFS study of the high-affinity copper-binding site of human PrP(91-231) and its low-resolution structure in solution. J Mol Biol. 2001 Aug 17;311(3):467–473. doi: 10.1006/jmbi.2001.4795. [DOI] [PubMed] [Google Scholar]
  16. Hornemann S., Glockshuber R. A scrapie-like unfolding intermediate of the prion protein domain PrP(121-231) induced by acidic pH. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6010–6014. doi: 10.1073/pnas.95.11.6010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hornemann S., Korth C., Oesch B., Riek R., Wider G., Wüthrich K., Glockshuber R. Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization. FEBS Lett. 1997 Aug 18;413(2):277–281. doi: 10.1016/s0014-5793(97)00921-6. [DOI] [PubMed] [Google Scholar]
  18. Hornshaw M. P., McDermott J. R., Candy J. M., Lakey J. H. Copper binding to the N-terminal tandem repeat region of mammalian and avian prion protein: structural studies using synthetic peptides. Biochem Biophys Res Commun. 1995 Sep 25;214(3):993–999. doi: 10.1006/bbrc.1995.2384. [DOI] [PubMed] [Google Scholar]
  19. Hosszu L. L., Baxter N. J., Jackson G. S., Power A., Clarke A. R., Waltho J. P., Craven C. J., Collinge J. Structural mobility of the human prion protein probed by backbone hydrogen exchange. Nat Struct Biol. 1999 Aug;6(8):740–743. doi: 10.1038/11507. [DOI] [PubMed] [Google Scholar]
  20. Houston Fiona, Goldmann Wilfred, Chong Angela, Jeffrey Martin, González Lorenzo, Foster James, Parnham David, Hunter Nora. Prion diseases: BSE in sheep bred for resistance to infection. Nature. 2003 May 29;423(6939):498–498. doi: 10.1038/423498a. [DOI] [PubMed] [Google Scholar]
  21. Jackson G. S., Hosszu L. L., Power A., Hill A. F., Kenney J., Saibil H., Craven C. J., Waltho J. P., Clarke A. R., Collinge J. Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science. 1999 Mar 19;283(5409):1935–1937. doi: 10.1126/science.283.5409.1935. [DOI] [PubMed] [Google Scholar]
  22. Jackson G. S., Murray I., Hosszu L. L., Gibbs N., Waltho J. P., Clarke A. R., Collinge J. Location and properties of metal-binding sites on the human prion protein. Proc Natl Acad Sci U S A. 2001 Jul 3;98(15):8531–8535. doi: 10.1073/pnas.151038498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kazlauskaite Jurate, Sanghera Narinder, Sylvester Ian, Vénien-Bryan Catherine, Pinheiro Teresa J. T. Structural changes of the prion protein in lipid membranes leading to aggregation and fibrillization. Biochemistry. 2003 Mar 25;42(11):3295–3304. doi: 10.1021/bi026872q. [DOI] [PubMed] [Google Scholar]
  24. Kramer M. L., Kratzin H. D., Schmidt B., Römer A., Windl O., Liemann S., Hornemann S., Kretzschmar H. Prion protein binds copper within the physiological concentration range. J Biol Chem. 2001 Feb 27;276(20):16711–16719. doi: 10.1074/jbc.M006554200. [DOI] [PubMed] [Google Scholar]
  25. Li R., Liu T., Wong B. S., Pan T., Morillas M., Swietnicki W., O'Rourke K., Gambetti P., Surewicz W. K., Sy M. S. Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus. J Mol Biol. 2000 Aug 18;301(3):567–573. doi: 10.1006/jmbi.2000.3986. [DOI] [PubMed] [Google Scholar]
  26. Liemann S., Glockshuber R. Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein. Biochemistry. 1999 Mar 16;38(11):3258–3267. doi: 10.1021/bi982714g. [DOI] [PubMed] [Google Scholar]
  27. Lu B. Y., Chang J. Y. Isolation of isoforms of mouse prion protein with PrP(SC)-like structural properties. Biochemistry. 2001 Nov 6;40(44):13390–13396. doi: 10.1021/bi011111t. [DOI] [PubMed] [Google Scholar]
  28. López Garcia F., Zahn R., Riek R., Wüthrich K. NMR structure of the bovine prion protein. Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8334–8339. doi: 10.1073/pnas.97.15.8334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McKenzie D., Bartz J., Mirwald J., Olander D., Marsh R., Aiken J. Reversibility of scrapie inactivation is enhanced by copper. J Biol Chem. 1998 Oct 2;273(40):25545–25547. doi: 10.1074/jbc.273.40.25545. [DOI] [PubMed] [Google Scholar]
  30. McKinley M. P., Meyer R. K., Kenaga L., Rahbar F., Cotter R., Serban A., Prusiner S. B. Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis. J Virol. 1991 Mar;65(3):1340–1351. doi: 10.1128/jvi.65.3.1340-1351.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miura T., Hori-i A., Mototani H., Takeuchi H. Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence. Biochemistry. 1999 Aug 31;38(35):11560–11569. doi: 10.1021/bi9909389. [DOI] [PubMed] [Google Scholar]
  32. Morillas M., Swietnicki W., Gambetti P., Surewicz W. K. Membrane environment alters the conformational structure of the recombinant human prion protein. J Biol Chem. 1999 Dec 24;274(52):36859–36865. doi: 10.1074/jbc.274.52.36859. [DOI] [PubMed] [Google Scholar]
  33. Morillas M., Vanik D. L., Surewicz W. K. On the mechanism of alpha-helix to beta-sheet transition in the recombinant prion protein. Biochemistry. 2001 Jun 12;40(23):6982–6987. doi: 10.1021/bi010232q. [DOI] [PubMed] [Google Scholar]
  34. Pan K. M., Baldwin M., Nguyen J., Gasset M., Serban A., Groth D., Mehlhorn I., Huang Z., Fletterick R. J., Cohen F. E. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10962–10966. doi: 10.1073/pnas.90.23.10962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pauly P. C., Harris D. A. Copper stimulates endocytosis of the prion protein. J Biol Chem. 1998 Dec 11;273(50):33107–33110. doi: 10.1074/jbc.273.50.33107. [DOI] [PubMed] [Google Scholar]
  36. Perrier Véronique, Kaneko Kiyotoshi, Safar Jiri, Vergara Julie, Tremblay Patrick, DeArmond Stephen J., Cohen Fred E., Prusiner Stanley B., Wallace Andrew C. Dominant-negative inhibition of prion replication in transgenic mice. Proc Natl Acad Sci U S A. 2002 Sep 23;99(20):13079–13084. doi: 10.1073/pnas.182425299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Provencher S. W., Glöckner J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry. 1981 Jan 6;20(1):33–37. doi: 10.1021/bi00504a006. [DOI] [PubMed] [Google Scholar]
  38. Prusiner S. B., McKinley M. P., Bowman K. A., Bolton D. C., Bendheim P. E., Groth D. F., Glenner G. G. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 1983 Dec;35(2 Pt 1):349–358. doi: 10.1016/0092-8674(83)90168-x. [DOI] [PubMed] [Google Scholar]
  39. Prusiner S. B. Novel proteinaceous infectious particles cause scrapie. Science. 1982 Apr 9;216(4542):136–144. doi: 10.1126/science.6801762. [DOI] [PubMed] [Google Scholar]
  40. Qin K., Yang D. S., Yang Y., Chishti M. A., Meng L. J., Kretzschmar H. A., Yip C. M., Fraser P. E., Westaway D. Copper(II)-induced conformational changes and protease resistance in recombinant and cellular PrP. Effect of protein age and deamidation. J Biol Chem. 2000 Jun 23;275(25):19121–19131. doi: 10.1074/jbc.275.25.19121. [DOI] [PubMed] [Google Scholar]
  41. Qin Kefeng, Yang Ying, Mastrangelo Peter, Westaway David. Mapping Cu(II) binding sites in prion proteins by diethyl pyrocarbonate modification and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric footprinting. J Biol Chem. 2001 Nov 6;277(3):1981–1990. doi: 10.1074/jbc.M108744200. [DOI] [PubMed] [Google Scholar]
  42. Quaglio E., Chiesa R., Harris D. A. Copper converts the cellular prion protein into a protease-resistant species that is distinct from the scrapie isoform. J Biol Chem. 2001 Jan 18;276(14):11432–11438. doi: 10.1074/jbc.M009666200. [DOI] [PubMed] [Google Scholar]
  43. Rezaei H., Marc D., Choiset Y., Takahashi M., Hui Bon Hoa G., Haertlé T., Grosclaude J., Debey P. High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility. Eur J Biochem. 2000 May;267(10):2833–2839. doi: 10.1046/j.1432-1033.2000.01347.x. [DOI] [PubMed] [Google Scholar]
  44. Rezaei Human, Choiset Yvan, Eghiaian Frederic, Treguer Eric, Mentre Pascale, Debey Pascale, Grosclaude Jeanne, Haertle Thomas. Amyloidogenic unfolding intermediates differentiate sheep prion protein variants. J Mol Biol. 2002 Sep 27;322(4):799–814. doi: 10.1016/s0022-2836(02)00856-2. [DOI] [PubMed] [Google Scholar]
  45. Riek R., Hornemann S., Wider G., Billeter M., Glockshuber R., Wüthrich K. NMR structure of the mouse prion protein domain PrP(121-231). Nature. 1996 Jul 11;382(6587):180–182. doi: 10.1038/382180a0. [DOI] [PubMed] [Google Scholar]
  46. Riek R., Hornemann S., Wider G., Glockshuber R., Wüthrich K. NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231). FEBS Lett. 1997 Aug 18;413(2):282–288. doi: 10.1016/s0014-5793(97)00920-4. [DOI] [PubMed] [Google Scholar]
  47. Speare Jonathan O., Rush Thomas S., 3rd, Bloom Marshall E., Caughey Byron. The role of helix 1 aspartates and salt bridges in the stability and conversion of prion protein. J Biol Chem. 2003 Jan 27;278(14):12522–12529. doi: 10.1074/jbc.M211599200. [DOI] [PubMed] [Google Scholar]
  48. Stahl N., Baldwin M. A., Teplow D. B., Hood L., Gibson B. W., Burlingame A. L., Prusiner S. B. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry. 1993 Mar 2;32(8):1991–2002. doi: 10.1021/bi00059a016. [DOI] [PubMed] [Google Scholar]
  49. Stöckel J., Safar J., Wallace A. C., Cohen F. E., Prusiner S. B. Prion protein selectively binds copper(II) ions. Biochemistry. 1998 May 19;37(20):7185–7193. doi: 10.1021/bi972827k. [DOI] [PubMed] [Google Scholar]
  50. Swietnicki W., Petersen R. B., Gambetti P., Surewicz W. K. Familial mutations and the thermodynamic stability of the recombinant human prion protein. J Biol Chem. 1998 Nov 20;273(47):31048–31052. doi: 10.1074/jbc.273.47.31048. [DOI] [PubMed] [Google Scholar]
  51. Swietnicki W., Petersen R., Gambetti P., Surewicz W. K. pH-dependent stability and conformation of the recombinant human prion protein PrP(90-231). J Biol Chem. 1997 Oct 31;272(44):27517–27520. doi: 10.1074/jbc.272.44.27517. [DOI] [PubMed] [Google Scholar]
  52. Thackray Alana M., Madec Jean-Yves, Wong Edmond, Morgan-Warren Robert, Brown David R., Baron Thierry, Bujdoso Raymond. Detection of bovine spongiform encephalopathy, ovine scrapie prion-related protein (PrPSc) and normal PrPc by monoclonal antibodies raised to copper-refolded prion protein. Biochem J. 2003 Feb 15;370(Pt 1):81–90. doi: 10.1042/BJ20021280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Viles J. H., Cohen F. E., Prusiner S. B., Goodin D. B., Wright P. E., Dyson H. J. Copper binding to the prion protein: structural implications of four identical cooperative binding sites. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2042–2047. doi: 10.1073/pnas.96.5.2042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wadsworth J. D., Hill A. F., Joiner S., Jackson G. S., Clarke A. R., Collinge J. Strain-specific prion-protein conformation determined by metal ions. Nat Cell Biol. 1999 May;1(1):55–59. doi: 10.1038/9030. [DOI] [PubMed] [Google Scholar]
  55. Whittal R. M., Ball H. L., Cohen F. E., Burlingame A. L., Prusiner S. B., Baldwin M. A. Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. Protein Sci. 2000 Feb;9(2):332–343. doi: 10.1110/ps.9.2.332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wildegger G., Liemann S., Glockshuber R. Extremely rapid folding of the C-terminal domain of the prion protein without kinetic intermediates. Nat Struct Biol. 1999 Jun;6(6):550–553. doi: 10.1038/9323. [DOI] [PubMed] [Google Scholar]
  57. Zahn R., Liu A., Lührs T., Riek R., von Schroetter C., López García F., Billeter M., Calzolai L., Wider G., Wüthrich K. NMR solution structure of the human prion protein. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):145–150. doi: 10.1073/pnas.97.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhang H., Stockel J., Mehlhorn I., Groth D., Baldwin M. A., Prusiner S. B., James T. L., Cohen F. E. Physical studies of conformational plasticity in a recombinant prion protein. Biochemistry. 1997 Mar 25;36(12):3543–3553. doi: 10.1021/bi961965r. [DOI] [PubMed] [Google Scholar]

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