Prion Protein Binds Copper within the Physiological Concentration Range

Abstract

The prion protein is known to be a copper-binding protein, but affinity and stoichiometry data for the full-length protein at a physiological pH of 7 were lacking. Furthermore, it was unknown whether only the highly flexible N-terminal segment with its octarepeat region is involved in copper binding or whether the structured C-terminal domain is also involved. Therefore we systematically investigated the stoichiometry and affinity of copper binding to full-length prion protein PrP23–231 and to different N- and C-terminal fragments using electrospray ionization mass spectrometry and fluorescence spectroscopy. Our data indicate that the unstructured N-terminal segment is the cooperative copper-binding domain of the prion protein. The prion protein binds up to five copper(II) ions with half-maximal binding at ∼2 μm. This argues strongly for a direct role of the prion protein in copper metabolism, since it is almost saturated at about 5 μm,and the exchangeable copper pool concentration in blood is about 8 μm. The prion protein is known to be a copper-binding protein, but affinity and stoichiometry data for the full-length protein at a physiological pH of 7 were lacking. Furthermore, it was unknown whether only the highly flexible N-terminal segment with its octarepeat region is involved in copper binding or whether the structured C-terminal domain is also involved. Therefore we systematically investigated the stoichiometry and affinity of copper binding to full-length prion protein PrP23–231 and to different N- and C-terminal fragments using electrospray ionization mass spectrometry and fluorescence spectroscopy. Our data indicate that the unstructured N-terminal segment is the cooperative copper-binding domain of the prion protein. The prion protein binds up to five copper(II) ions with half-maximal binding at ∼2 μm. This argues strongly for a direct role of the prion protein in copper metabolism, since it is almost saturated at about 5 μm,and the exchangeable copper pool concentration in blood is about 8 μm. prion protein electrospray ionization 4-morpholinepropanesulfonic acid dodecyltrimethylammonium chloride N-ethylmorpholine high performance liquid chromatography average mass unit mass spectrometry Prion diseases are fatal neurodegenerative diseases thought to be caused by conformational transition of the native and predominantly α-helical prion protein (PrPC)1 to the significantly more β-sheet-containing pathogenic isoform (PrPSc) (1Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Ziwei H. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2076) Google Scholar). This conformational transition apparently induces the formation of PrPSc aggregates (2Post K. Pitschke M. Schäfer O. Wille H. Appel T.R. Kirsch D. Mehlhorn I. Serban H. Prusiner S.B. Riesner D. Biol. Chem. 1998; 379: 1307-1317Crossref PubMed Scopus (85) Google Scholar), which are, in contrast to PrPC, highly protease-resistant (3McKinley M.P. Bolton D.C. Prusiner S.B. Cell. 1983; 35: 57-62Abstract Full Text PDF PubMed Scopus (719) Google Scholar). During infection, PrPSc appears to serve as a template for the conversion of the native prion protein, because host PrPC expression is required for infection with prions (4Büeler H. Fischer M. Lang Y. Bluethmann H. Lipp H.-P. DeArmond S.J. Prusiner S.B. Auet M. Weissmann C. Nature. 1992; 356: 577-582Crossref PubMed Scopus (1440) Google Scholar). There is evidence that copper plays a role in the formation of PrPSc (5McKenzie D. Bartz J. Mirwald J. Olander D. Marsh R. Aiken J. J. Biol. Chem. 1998; 273: 25545-25547Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). PrPC is nearly ubiquitously distributed to all tissues. The highest levels are found in the brain (6Bendheim P.E. Brown H.R. Rudelli R.D. Scala L.J. Goller N.L. Wen G.Y. Kascsak R.J. Cashman N.R. Bolton D.C. Neurology. 1992; 42: 149-156Crossref PubMed Google Scholar). Native PrPC is an asparagine-linked sialoglycoprotein that is attached to the surface of the plasma membrane via a C-terminal glycophosphatidylinositol anchor (7Stahl N. Borchelt D.R. Prusiner S.B. Biochemistry. 1990; 29: 5405-5412Crossref PubMed Scopus (225) Google Scholar). The structured C-terminal half of the full-length recombinant PrP23–231 (amino acids 23–231) from amino acids 126–231 is made up of two strands of one small antiparallel β-sheet and three α-helices (8Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wüthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1129) Google Scholar). The N-terminal half from amino acids 23–125 showed no structure in NMR analysis and is therefore postulated to be highly flexible (9Riek R. Hornemann S. Wider G. Glockshuber R. Wüthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (664) Google Scholar, 10Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar). Although many efforts have been made the physiological function of PrPC has not yet been identified (11Büeler H. Aguzzi A. Sailer A. Greiner R.-A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1810) Google Scholar, 12Lledo P.M. Tremblay P. DeArmond S.J. Prusiner S.B. Nicoll R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2403-3407Crossref PubMed Scopus (191) Google Scholar). In 1995 it was suggested that the octarepeat region ([PHGGGWGQ]4) from amino acids 60–91 within the flexible N-terminal half of the prion protein may play a role in binding copper (13Hornshaw M.P. McDermott J.R. Candy J.M. Biochem. Biophys. Res. Commun. 1995; 207: 621-629Crossref PubMed Scopus (322) Google Scholar). We have shown that PrPC has indeed a function in synaptic copper binding (14Herms J. Tings T. Gall S. Madlung A. Giese A. Siebert H. Schürrmann P. Windl O. Brose N. Kretzschmar H. J. Neurosci. 1999; 19: 8866-8875Crossref PubMed Google Scholar). Heavy metal binding studies indicated that PrPC seems to specifically bind copper (15Stöckel J. Safar J. Wallace A.C. Cohen F.E. Prusiner S.B. Biochemistry. 1998; 37: 7185-7193Crossref PubMed Scopus (494) Google Scholar). Half-maximal binding of two copper ions at 14 μm was reported for Syrian hamsterShamPrP29–231 at pH 6.0 (15Stöckel J. Safar J. Wallace A.C. Cohen F.E. Prusiner S.B. Biochemistry. 1998; 37: 7185-7193Crossref PubMed Scopus (494) Google Scholar). In contrast, binding of 4 and 5.6 copper ions to the octarepeat region peptide (16Hornshaw M.P. McDermott J.R. Candy J.M. Lakey J.H. Biochem. Biophys. Res. Commun. 1995; 214: 993-999Crossref PubMed Scopus (295) Google Scholar,17Viles J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar) and humPrP23–98 (18Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar) at pH 7.4 with half-maximal binding at about 6 μm were found, respectively. The copper binding to the octarepeat region was shown to be strongly pH-dependent in the range from 5 to 7 (14Herms J. Tings T. Gall S. Madlung A. Giese A. Siebert H. Schürrmann P. Windl O. Brose N. Kretzschmar H. J. Neurosci. 1999; 19: 8866-8875Crossref PubMed Google Scholar) with almost no binding at pH 5. The lack of affinity and stoichiometry data for the full-length prion protein at a physiological pH of 7, as well as the question of which part of the prion protein is responsible for the copper binding, prompted us to systematically investigate the copper binding to the full-length prion protein as well as to different N- and C-terminal fragments using fluorescence spectroscopy and electrospray ionization mass spectrometry. In this paper we present for the first time copper binding data on the full-length prion protein under physiological conditions at pH 7. Our data show that the prion protein binds up to five copper ions and is almost saturated at 5 μm copper(II). Furthermore, our data suggest that the highly flexible N-terminal half is the cooperative copper-binding domain of the prion protein. MOPS, dodecyltrimethylammonium chloride, CuSO4·5H2O, andN-ethylmorpholine were from was from and were from were of The and on an using amino acids with and with J. D. Lett. 1990; Scopus Google Scholar). from and of were first by using a with a from to acid for were on an with a from to pH were by on a using a of The N-terminal humPrP23–98 was in to 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. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar). expression and of the protein, the was to an was with a from up to pH The was for the expression of the of the from O. M. R. R. T. R. A. J. 1996; PubMed Scopus Google Scholar). The region of the N-terminal segment of to was via under conditions using and a and a two and an The was the of the expression and the of the was by analysis The expression of the in was with were with and at for The was to an with pH and with a up to pH 6.0 from the N-terminal fragments were by on a using a of to The full-length was and to S. Glockshuber R. Biochemistry. 1999; PubMed Scopus (295) Google Scholar). protein was with to of The C-terminal was and as S. Glockshuber R. J. Biol. 1996; Scopus Google Scholar). spectroscopy was on an from were in MOPS, pH and was with to of metal a of was and the fluorescence was on the at from to 5 and was in the range of of the for was to was to by in the in the fluorescence the average of at three were at to of copper in the fluorescence were for Biochem. 1983; PubMed Scopus Google Scholar). were for spectrometry of N-terminal fragments with and copper were on a in the The was acid at pH The was was to and protein were in 6 chloride to and von von Biochem. PubMed Scopus Google Scholar). by amino acid analysis for in mass spectrometry was to the stoichiometry of copper binding to the different N-terminal as well as for the C-terminal and for the full-length prion protein The conditions for metal studies at pH 7.4 with to and were we N-ethylmorpholine at pH 7.4 as a for metal studies with because it not with copper binding to and protein with Furthermore, of or significantly the copper in the mass not and were therefore binding the copper concentration as well as the peptide concentration were as as a we since it is to the prion protein with to its and the copper binding at the N-terminal amino as well as C-terminal and it has one at amino acid the first which is to the shown in the of with and and copper at a of copper(II) no binding the first and copper mass of and average mass were respectively. The copper binding to as a that is an for copper with at a physiological pH of 7. mass spectrometry of the N-terminal fragments in the average for the In the of a with with a mass of is The mass of a or in the The mass from the of was the This is to the of to P. R. P. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar). The for the for which a mass of was The mass indicated that the three C-terminal amino acids have been for the copper binding of the prion protein and its fragments from mass in a a of copper(II) to the in mass with copper of μm copper to μm to up to copper(II) peptide The two small the two copper are to and in mass spectrometry of and at μm copper(II) to μm in the of to the formation of up to five copper that the apparently to and one copper are also the for the with two and three because only by 4 and as of μm copper(II) to μm humPrP23–98 to the of for up to copper be that a not all significantly the of for copper a copper binding In contrast, only for up to five copper were on of μm copper to μm of the 14 amino acids The of to and on of copper conformational with copper binding, in a more or structured with the The structured C-terminal domain of the prion was by mass spectrometry and a mass of which to the average of of up to μm copper to only one copper was This be from the This appears to be of because at μm which is the in only a of is μm copper of the almost and the for one copper This almost the copper concentration up to μm and that is one copper-binding with affinity in In contrast to the N-terminal is no of the to no conformational on binding of the copper the mass of the an average mass of with a of is which is about 6 the mass of The for this was of up to μm not well copper to the and the high the copper be from the The showed for up to five copper on of μm In contrast to the and are only in the from to a of The average for all and are in I. this the average mass the copper almost to which is in copper(II) binding mass spectrometry the average mass of copper is the of is as of two on binding of copper the of only an copper appears to be which is almost but only five copper were which an almost C-terminal to the octarepeat binds only five copper Therefore the binding of a copper appears to be an copper-binding up to five copper ions to be specifically to copper ions are to the octarepeat an copper-binding within the N-terminal the copper-binding which is this binding has to be of the octarepeat and of the copper binding to PrPC and its fragments were with to to binding of copper to PrPC and its was to and to was at a concentration concentration PubMed Scopus Google Scholar). for the of the peptide or PrPC were to be significantly the to that the copper concentration is to the copper in contrast the fluorescence of and is not only by conformational by copper(II) binding but also by of copper(II) with and C. T. Biophys. PubMed Scopus Google Scholar, H. Scholar, Biochem. PubMed Scopus Google Scholar). fluorescence have to be at copper the to data from the fluorescence The fluorescence with copper(II) for the N-terminal fragments the structured C-terminal as well as for the full-length recombinant are shown in that for show more or in fluorescence of a cooperative binding appears that the be saturated with copper at high This is to which be shown by to the H. Scholar), is the fluorescence at the concentration of the is copper in this is the fluorescence and is the also the the of copper binding to the or protein, the be in this is only to with the of 4 the in the of PrPC and its N-terminal fragments showed that the fluorescence μm are the of The of the be by the different of of for of The fluorescence which has and only not only the fluorescence but also the fluorescence of which are to have different the as well as the of conformational fluorescence caused by binding of copper are by analysis at high copper this has to be because it is only In the of conformational fluorescence for be a to is the of conformational fluorescence to the the from the have to be by the of the The and The is to fluorescence data by to copper binding The for a for the of from almost to about that the more in the with The for the at for is not known but has to be an of the C-terminal Although the of 4 not be well with a of be only data from to μm for the and conformational fluorescence of the prion protein and its N-terminal fragments by by with by to data in are the of by to by to by to by to by to cooperative were and a for an cooperative were and a for an by with by to data in are the of by to cooperative were and a for an in a the fluorescence it is that is a in affinity for copper(II) from to a small in affinity be to humPrP23–98 the 4 as well as for the full-length recombinant 4 to be copper binding data the were by mass spectrometry has that copper ions bind to the octarepeat region The mass spectrometry data suggest that one copper binding This was to be because the only be with a for an binding almost fluorescence cooperative binding data of were to is the and are the of fluorescence and the for the binding and are the fluorescence the and the for the cooperative binding is the and is the copper the concentration was to be significantly the the or copper concentration was to be to the copper the the from were for in to all fluorescence because only cooperative binding were and the for the was The for was to because mass spectrometry suggested binding of only one copper and no cooperative binding The data show that and N-terminal in the of the octarepeat have a on and The for the cooperative binding of copper ions to the octarepeat region significantly from to μm and only to μm for significantly from to from to almost for the cooperative copper-binding in the full-length prion protein. in the are for the binding the significantly from μm to and μm the one copper-binding in a of about 8 μm was that no was we present for the first time affinity and stoichiometry data on the copper binding to and the full-length prion protein under physiological conditions as well as for a of the prion protein D. J. PubMed Google Scholar, D.B. P. P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Our that the full-length prion protein binds copper within the physiological concentration range with binding of five copper ions Furthermore, we that the of copper binding is not to N-terminal fragments but is a of the full-length protein. The of the data for full-length with and N-terminal fragments that the N-terminal segment the cooperative copper-binding domain of the prion protein. spectrometry has as a in the and of copper and metal binding stoichiometry of and 1997; PubMed Scopus Google Scholar, M. M. Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). In contrast to Cohen F.E. Prusiner S.B. Baldwin Sci. PubMed Scopus Google Scholar) we only for the copper binding stoichiometry studies for our fluorescence affinity data indicated the copper binding of the prion protein and its fragments to be almost saturated with copper under the in it is to mass predominantly the or five copper are This is not the as from the be by the high of the N-terminal copper binding domain and its to the more copper binding in which the copper binding are by structure Chem. 42: PubMed Google Scholar). the mass not to the in but for the prion protein and its showed that are to of and The of on the mass of with as is shown in 5. In contrast to the the and copper-binding are significantly using the N-ethylmorpholine the of with a copper binding to in spectroscopy J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar). mass spectrometry of copper binding to prion protein and appears to be to the and Therefore we have N-ethylmorpholine as in mass spectrometry for metal studies at a physiological pH of 7. Our that the octarepeat region binds copper ions mass spectrometry (13Hornshaw M.P. McDermott J.R. Candy J.M. Biochem. Biophys. Res. Commun. 1995; 207: 621-629Crossref PubMed Scopus (322) Google Scholar, Cohen F.E. Prusiner S.B. Baldwin Sci. PubMed Scopus Google Scholar) and spectroscopy data J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar). The mass of one copper-binding This is in with the binding of 5.6 copper(II) ions to humPrP23–98 from our (18Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar). as well as from the mass a binding which was also Cohen F.E. Prusiner S.B. Baldwin Sci. PubMed Scopus Google Scholar), be because only five copper ions were this has to be The of the N-terminal amino for the was by N-terminal Cohen F.E. Prusiner S.B. Baldwin Sci. PubMed Scopus Google Scholar). spectrometry data showed binding of up to five copper ions to the full-length protein, one copper-binding with the of the octarepeat The data for the structured C-terminal domain three one copper-binding with an of 8 μm. This affinity is to be of physiological copper in the at two are from which one has to be a or I. K. in Scholar). for this in the NMR structure of the C-terminal we found a made up of and are on the surface and to In contrast to the C-terminal for the binding in and full-length prion protein, we a of about μm. the copper is it has to C-terminal of the octarepeat This binding be at because it is known that the the is the copper-binding in and I. K. in Scholar). In structured at two two are for copper binding in the concentration range Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). In flexible structure only one is for the copper In the peptide in which peptide is involved as a in copper binding Biophys. 1993; PubMed Scopus Google Scholar). of peptide in the C-terminal but the of peptide as I. K. in Scholar). In this the of the three with the in a copper The at is but also be involved in copper are required to the role of or in the copper binding of prion protein. The of fluorescence for the prion protein and its N-terminal fragments indicated an cooperative binding This well with spectroscopy data for which is almost to our octarepeat J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar). In contrast, Cohen F.E. Prusiner S.B. Baldwin Sci. PubMed Scopus Google Scholar) have a This is by the of mass spectrometry in analysis and the conditions as Our that the cooperative copper binding is not to the octarepeat but is a of the full-length prion protein. The fluorescence data for a cooperative of which is almost to the of 6 μm by spectroscopy J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar). with with a of μm (18Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar), we have significantly of and μm for the cooperative and the respectively. This be by the different Our affinity data for are to with by Cohen F.E. Prusiner S.B. Baldwin Sci. PubMed Scopus Google Scholar) for the and because we have one cooperative of from which only two were in the Cohen F.E. Prusiner S.B. Baldwin Sci. PubMed Scopus Google Scholar). The of the fluorescence with the full-length prion as well as the data indicated that the N-terminal segment is a copper-binding The affinity copper-binding in the C-terminal domain apparently no on the copper-binding and affinity of the N-terminal Although the N-terminal segment up to amino acid is highly and N-terminal of the octarepeat region have a on the affinity and of the and cooperative binding respectively. the octarepeat region is to The affinity of the significantly on N-terminal the fluorescence data of the cooperative as well as for the indicated that are the data show that the is almost saturated at about 5 μm copper(II). This is within the physiological concentration range of the copper concentration in blood plasma of μm M. J. 1996; Google Scholar). this concentration not the copper because about μm copper is to in a In contrast about μm is to μm to and μm is to amino acids in an exchangeable only the copper pool of μm by and the copper of the prion protein, is for a direct role of the prion protein in copper copper from to μm are found in the μm is present in the synaptic A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In contrast to blood plasma the conditions at the synaptic and in the are different in that no or is The cooperative copper-binding of the prion protein within the physiological concentration range two to the cooperative binding of it play a role in copper the prion protein be a copper to the protein which also binds in a cooperative In is known about the involved in copper and the by which copper is the plasma membrane S. 1995; PubMed Scopus Google Scholar). copper by the prion protein be to which is by and prion protein and copper at an pH of (15Stöckel J. Safar J. Wallace A.C. Cohen F.E. Prusiner S.B. Biochemistry. 1998; 37: 7185-7193Crossref PubMed Scopus (494) Google Scholar). function for PrPC is by the of with copper J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), the pH-dependent copper binding J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar), and the copper of brain in (18Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar). This was by our that PrPC is apparently in and that the of PrPC in strongly the copper of (14Herms J. Tings T. Gall S. Madlung A. Giese A. Siebert H. Schürrmann P. Windl O. Brose N. Kretzschmar H. J. Neurosci. 1999; 19: 8866-8875Crossref PubMed Google Scholar). This that PrPC is involved in the synaptic copper The in binding of copper to the prion protein is not yet PrPC not bind copper under it serve as an copper is known that and plasma copper in blood M. J. 1996; Google Scholar). it be that PrPC binds copper and on copper binding to appears to play a role in the prion as well S. J. Biol. 1999; PubMed Scopus Google Scholar). apparently because it the of PrPSc (5McKenzie D. Bartz J. Mirwald J. Olander D. Marsh R. Aiken J. J. Biol. Chem. 1998; 273: 25545-25547Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our of a affinity copper-binding in the C-terminal domain of PrPC be for the conformational to are to the of the copper binding in the octarepeat region as well as to the role of copper binding for the prion protein in We for the for the of and K. for the of fluorescence Furthermore, we for in expression and for