Copper Binding to the Amyloid-β (Aβ) Peptide Associated with Alzheimer's Disease
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m313572200
ISSN1083-351X
AutoresChristopher D. Syme, Rebecca C. Nadal, Stephen E. J. Rigby, John H. Viles,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoThere is now direct evidence that copper is bound to amyloid-β peptide (Aβ) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of Aβ and free radical damage, and Cu2+ chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu2+ to Aβ in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of Aβ. Competition studies with glycine and l-histidine indicate that copper binds to Aβ-(1–28) at pH 7.4 with an affinity of Ka ∼107m–1. 1H NMR indicates that histidine residues are involved in Cu2+ coordination but that Tyr10 is not. Studies using analogues of Aβ-(1–28) in which each of the histidine residues have been replaced by alanine or in which the N terminus is acetylated suggest that the N terminus and His13 are crucial for Cu2+ binding and that His6 and His14 are also implicated. Evidence for the link between Alzheimer's disease and Cu2+ is growing, and our studies have made a significant contribution to understanding the mode of Cu2+ binding to Aβ in solution. There is now direct evidence that copper is bound to amyloid-β peptide (Aβ) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of Aβ and free radical damage, and Cu2+ chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu2+ to Aβ in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of Aβ. Competition studies with glycine and l-histidine indicate that copper binds to Aβ-(1–28) at pH 7.4 with an affinity of Ka ∼107m–1. 1H NMR indicates that histidine residues are involved in Cu2+ coordination but that Tyr10 is not. Studies using analogues of Aβ-(1–28) in which each of the histidine residues have been replaced by alanine or in which the N terminus is acetylated suggest that the N terminus and His13 are crucial for Cu2+ binding and that His6 and His14 are also implicated. Evidence for the link between Alzheimer's disease and Cu2+ is growing, and our studies have made a significant contribution to understanding the mode of Cu2+ binding to Aβ in solution. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid-β peptide; EPR, electron paramagnetic resonance. 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid-β peptide; EPR, electron paramagnetic resonance. is characterized by innumerable deposits of extracellular amyloid plaques. A small peptide, amyloid-β peptide (Aβ), plays a critical role in the initial build up of these amyloid plaques and is the main constituent of the amyloid deposits (1Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Google Scholar, 2Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Google Scholar). In addition, genetic alterations underlying familial AD are associated with an increase in the production and/or the deposition of Aβ in the brain (3Roses A.D. Curr. Opin. Neurobiol. 1996; 6: 644-650Google Scholar, 4Selkoe D.J. Science. 1997; 275: 630-631Google Scholar, 5Selkoe D.J. J. Biol. Chem. 1996; 271: 18295-18298Google Scholar, 6Mills J. Reiner P.B. J. Neurochem. 1999; 72: 443-460Google Scholar). Amyloid-β peptide can be between 39 and 43 residues in length, of which Aβ-(1–40) and Aβ-(1–42) are the most abundant fragments. The N-terminal portion of Aβ is hydrophilic, whereas the C terminus amino acids 29–42 are rich in hydrophobic residues and represent the transmembrane region in the amyloid precursor protein. The sequence of human Aβ-(1–42) is as follows: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA. Soluble Aβ-(1–40) and Aβ-(1–42) are found in the cerebrospinal fluid and blood plasma of all humans where Aβ-(1–40) has a concentration of 5 nm in cerebrospinal fluid (7Lambert M.P. Barlow A.K. Chromy B.A. Edwards C. Freed R. Liosatos M. Morgan T.E. Rozovsky I. Trommer B. Viola K.L. Wals P. Zhang C. Finch C.E. Krafft G.A. Klein W.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6448-6453Google Scholar, 8Vigo-Pelfrey C. Lee D. Keim P. Lieberburg I. Schenk D.B. J. Neurochem. 1993; 61: 1965-1968Google Scholar). It is yet to be established what triggers Aβ to convert from its soluble form to an amyloidogenic form, but it has been shown that physiological levels of Cu2+ and Zn2+ cause marked aggregation of Aβ. This process is thought to be the prelude to amyloid formation (9Bush A.I. Pettingell W.H. Multhaup G. Paradis M.D. Vonsattel J.P. Gusella J.F. Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Google Scholar). Levels of these metals are elevated in amyloid plaque deposits: 0.4 mm and 1 mm for Cu2+ and Zn2+, respectively (10Lovell M.A. Robertson J.D. Teesdale W.J. Campbell J.L. Markesbery W.R. J. Neurol. Sci. 1998; 158: 47-52Google Scholar). Cu2+-induced aggregation of Aβ occurs as the pH is lowered to 6.8. This mildly acidic environment mimics a feature of inflammation found in AD (11Atwood C.S. Moir R.D. Huang X. Scarpa R.C. Bacarra N.M. Romano D.M. Hartshorn M.A. Tanzi R.E. Bush A.I. J. Biol. Chem. 1998; 273: 12817-12826Google Scholar). Studies on cerebrospinal fluid indicate that zinc will cause the selective aggregation of endogenous soluble Aβ peptide (12Brown A.M. Tummolo D.M. Rhodes K.J. Hofmann J.R. Jacobsen J.S. Sonnenberg-Reines J. J. Neurochem. 1997; 69: 1204-1212Google Scholar). Metal chelators specific to Cu2+ and Zn2+ will reverse this aggregation process (13Huang X. Atwood C.S. Moir R.D. Hartshorn M.A. Vonsattel J.P. Tanzi R.E. Bush A.I. J. Biol. Chem. 1997; 272: 26464-26470Google Scholar, 14Cherny R.A. Legg J.T. McLean C.A. Fairlie D.P. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; 274: 23223-23228Google Scholar). In addition, the neurotoxicity of Aβ is linked to metal-induced oxidative damage and is a feature of the pathogenesis of AD (15Smith M.A. Harris P.L. Sayre L.M. Perry G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9866-9868Google Scholar). A dual role as a pro- and antioxidant in copper redox cycling in a Fenton-type reaction has been proposed for Aβ peptide (16Kontush A. Free Radic. Biol. Med. 2001; 31: 1120-1131Google Scholar, 17Huang X. Atwood C.S. Hartshorn M.A. Multhaup G. Goldstein L.E. Scarpa R.C. Cuajungco M.P. Gray D.N. Lim J. Moir R.D. Tanzi R.E. Bush A.I. Biochemistry. 1999; 38: 7609-7616Google Scholar, 18Huang X. Cuajungco M.P. Atwood C.S. Hartshorn M.A. Tyndall J.D. Hanson G.R. Stokes K.C. Leopold M. Multhaup G. Goldstein L.E. Scarpa R.C. Saunders A.J. Lim J. Moir R.D. Glabe C. Bowden E.F. Masters C.L. Fairlie D.P. Tanzi R.E. Bush A.I. J. Biol. Chem. 1999; 274: 37111-37116Google Scholar, 19Atwood C.S. Huang X. Moir R.D. Tanzi R.E. Bush A.I. Met. Ions Biol. Syst. 1999; 36: 309-364Google Scholar, 20Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Google Scholar, 21Bondy S.C. Guo-Ross S.X. Truong A.T. Brain Res. 1998; 799: 91-96Google Scholar, 22Dikalov S.I. Vitek M.P. Maples K.R. Mason R.P. J. Biol. Chem. 1999; 274: 9392-9399Google Scholar). There are two commonly expressed objections to the role of copper in AD. The first objection is that AD is not associated with elevated exposure to environmental copper. It is important to clarify this misconception. The total concentration of copper within the brain is potentially more than sufficient to be neurotoxic. As a consequence, the brain has efficient homeostatic mechanisms in place to maintain compartmentalization of metal ions, which when compromised cause neurodegenerative diseases such as Wilson's and Menkes' disease. There is evidence to suggest that homeostatic mechanisms for metal ions are impaired in AD patients, and AD is characterized by altered metal ion-dependent processes and metal ion concentrations in the brain (23Cuajungco M.P. Lees G.J. Brain Res. Brain Res. Rev. 1997; 23: 219-236Google Scholar). The second objection is that the affinity of Aβ for Cu2+ is too low to bind these metals at their extracellular concentrations. This is also a misconception, since extracellular levels of Cu2+ may reach as high as 15 μm (10Lovell M.A. Robertson J.D. Teesdale W.J. Campbell J.L. Markesbery W.R. J. Neurol. Sci. 1998; 158: 47-52Google Scholar), whereas we show here that Aβ affinity for Cu2+ is at the submicromolar level and is reported to be much higher in amyloid plaques (24Atwood C.S. Scarpa R.C. Huang X. Moir R.D. Jones W.D. Fairlie D.P. Tanzi R.E. Bush A.I. J. Neurochem. 2000; 75: 1219-1233Google Scholar). A recent study using Raman spectroscopy has provided direct evidence that copper and zinc are bound via the histidine imidazole rings in isolated senile plaque cores (25Dong J. Atwood C.S. Anderson V.E. Siedlak S.L. Smith M.A. Perry G. Carey P.R. Biochemistry. 2003; 42: 2768-2773Google Scholar). Perhaps one of the most significant pieces of evidence to link copper with AD is the observation that normally insoluble amyloid deposits of postmortem brain tissue from AD patients can be solubilized in aqueous media by the presence of metal chelators specific to Cu2+ (14Cherny R.A. Legg J.T. McLean C.A. Fairlie D.P. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; 274: 23223-23228Google Scholar). It has been shown that the use of copper chelators can markedly inhibit amyloid accumulation in AD transgenic mice and are in trials as potential drug therapies for AD (26Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Google Scholar, 27Bush A.I. Neurobiol. Aging. 2002; 23: 1031-1038Google Scholar). A recent study has shown that trace amounts of copper in the drinking water of rabbits induce β-amyloid plaque accumulation (28Sparks D.L. Schreurs B.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11065-11069Google Scholar, 29Marx J. Science. 2003; 301: 905Google Scholar). Despite an increasing body of evidence to link Cu2+ with AD, the precise coordination geometry and the residues involved in Cu2+ ligation are yet to be established. Both monomeric and dimeric species have been proposed (30Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Google Scholar). In addition, there are disagreements as to the affinity and stoichiometry of binding. For example, both attomolar affinities (24Atwood C.S. Scarpa R.C. Huang X. Moir R.D. Jones W.D. Fairlie D.P. Tanzi R.E. Bush A.I. J. Neurochem. 2000; 75: 1219-1233Google Scholar) and micromolar affinities (31Garzon-Rodriguez W. Yatsimirsky A.K. Glabe C.G. Bioorg. Med. Chem. Lett. 1999; 9: 2243-2248Google Scholar) for Cu-Aβ-(1–42) have been reported. In this study, we use a range of complementary spectroscopies to characterize the binding of Cu2+ to Aβ and the structural changes induced in Aβ upon copper coordination. To facilitate solution spectroscopy methods, we have used the more soluble fragment of Aβ-(1–28), which lacks the C-terminal third of the molecule. Residues 29–42 are highly hydrophobic and are not believed to be associated with direct coordination of the metal ion (30Curtain C.C. Ali F. Volitakis I. Cherny R.A. Norton R.S. Beyreuther K. Barrow C.J. Masters C.L. Bush A.I. Barnham K.J. J. Biol. Chem. 2001; 276: 20466-20473Google Scholar, 41Miura T. Suzuki K. Kohata N. Takeuchi H. Biochemistry. 2000; 39: 7024-7031Google Scholar). In addition, we have studied a number of analogues of Aβ-(1–28) in which each of the three histidine residues have been replaced with an alanine. Peptide Synthesis and Purification—Peptides representing various fragments of the amyloid-β peptide were synthesized by employing solid phase Fmoc chemistry and produced by the ABC facility at Imperial College (London, UK). After removal from the resin and deprotection, the samples were purified using reverse phase high pressure liquid chromatography and characterized using mass spectrometry and 1H NMR. Titrations—The pH was measured before and after each spectrum was recorded. N-Ethylmorpholine buffer was found not to interfere with Cu2+ binding. Typically, 50 mm N-ethylmorpholine buffer was used for electron paramagnetic resonance (EPR) studies, whereas for 1H NMR and CD studies, samples were prepared in ultrahigh quality (>18 megaohms/cm resistivity) water, and the pH was adjusted using small amounts of 0.1 m NaOH or HCl. The peptide concentrations were determined using the extinction coefficient of 1280 m–1 cm–1 (due to the single tyrosine residue) (32Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Google Scholar). Typically, the freeze-dried peptides contained 5–10% moisture by weight. The addition of metal ions or competing ligands to the Aβ peptides was performed using small aliquots from stock aqueous solutions. Circular Dichroism—CD spectra were recorded on an AVIV Circular Dichroism model 202 spectrometer at 25 °C. Typically, a cell with a 0.1-cm path length was used for spectra recorded between 185 and 260 nm with sampling points every 0.5 nm. A 1-cm cell path length was used for data between 240 and 800 nm with a 2-nm sampling interval. A minimum of three scans were recorded, and base-line spectra were subtracted from each spectrum. AVIV software was used to smooth data when necessary. Data was processed using Kaleidagraph spreadsheet/graph package. Direct CD measurements (θ, in millidegrees) were converted to molar ellipticity, Δϵ (m–1 cm–1) using the relationship Δϵ = θ/33,000 × c × l, where c represents the concentration and l is the path length. Absorption Spectroscopy (UV-visible)—UV-visible electronic absorption spectra were obtained with a Hitachi U-3010 double beam spectrophotometer, using a 1-cm path length quartz cuvette. Fluorescence Spectroscopy—Fluorescence spectra were collected using a Hitachi F-2500 fluorescence spectrophotometer. An excitation frequency of 280 nm was used, and data were collected over the range of 290–400 nm. Samples were placed in a four-sided quartz fluorescence cuvette (Hellma), and data were recorded at room temperature. Stability Constants—The absolute affinity (pH-independent) of l-histidine for Cu2+ is 1.5 × 1010m–1; the apparent affinity at pH 7.8 is therefore 2.6 × 108m–1 or a dissociation constant of 1.5 nm (since log α = pKa – pH = 9.17 – 7.8 = 1.37 and log K1 (app) = log K1 – log α = 10.2 – 1.37 = 8.83 or 6.7 × 108m–1). Similarly, the absolute affinity (pH-independent) of glycine for Cu2+ is 1.2 × 109m–1; the apparent affinity at pH 7.8 is therefore 1.8 × 106m–1 or a dissociation constant of 500 nm (33Dawson R.M.C. Elliot D.C. Elliot W.H. Jones K.M. Data for Biochemical Research. Claredon Press, Oxford, UK1986Google Scholar). EPR—X-band EPR data were recorded using a Bruker ELEXSYS 500 spectrometer, operating at a microwave frequency of ∼9.3 GHz. All spectra were recorded using a microwave power of 0.63 mW across a sweep width of 2000 G (centered at 3200 G) with a modulation amplitude of 10 G. Samples were frozen in quartz tubes, and experiments were carried out at between 20 and 120 K using a liquid helium cryostat. A minimum of two scans were recorded per spectrum. All EPR spectra shown have been background-subtracted from a water blank with subsequent base-line correction in the XEPR software package using 3rd or 4th order polynomial splines. In order to analyze EPR data using the method described by Peisach and Blumberg (34Peisach J. Blumberg W.E. Arch. Biochem. Biophys. 1974; 165: 691-708Google Scholar), it is necessary to convert values from gauss (G) to millikaisers by using the formula AII (millikaisers) = 0.046686 × g × ΔH, where g = 2.0023 and ΔH is the AII splitting measured in gauss. NMR—Proton NMR data were collected using a Varian UNITYplus 600-MHz 1H frequency spectrometer. Data were processed using the VNMR software package. All spectra were recorded at 25 °C in 100% D2O solution, typically at a peptide concentration of 1 mm. Proton peak assignments were made by the analysis of two-dimensional total correlation spectroscopy and ROESY spectra of the apo peptides. Total correlation spectroscopy and ROESY spectra were collected with typical mixing times of ∼75 and ∼300 ms, respectively. Design of Peptides—Typically, the N terminus was left as the native amino group, whereas the truncated C terminus was blocked as the ethyl ester at the C terminus. Peptides synthesized of the human sequence are shown in Table I.Table IPeptides synthesizedDesignationSequenceAβ-(1-28)DAEFRHDSGYEVHHQKLVFFAEDVGSNKAβ(H6A)DAEFRADSGYEVHHQKLVFFAEDVGSNKAβ(H13A)DAEFRHDSGYEVAHQKLVFFAEDVGSNKAβ(H14A)DAEFRHDSGYEVHAQKLVFFAEDVGSNKAβ(Ac1-28)Ac—DAEFRHDSGYEVHHQKLVFFAEDVGSNKAβ-(1-16)DAEFRHDSGYEVHHQKAβ-(Ac10-16)Ac—YEVHHQKAβ-(1-11)DAEFRHDSGYE Open table in a new tab pH Dependence of Cu2+ Binding—Fig. 1 shows the EPR spectra of 0.8 mol eq of Cu2+ bound to Aβ-(1–28) over a range of pH values between 5 and 10. The EPR spectrum at pH 5 gives a single set of signals typical of type II copper, axial (square-planar or square-pyramidal) coordination geometry. The AII, gII, and g⊥ values are 177 G (∼16.5 millikaisers), 2.26, and 2.06, respectively. As the pH is increased, a new set of hyperfine peaks are observed to higher field with AII, gII, and g⊥ values of 170 G (∼15.9 millikaisers), 2.22, and 2.06, respectively. Commensurate with the appearance of a new set of axial signals, the spectra observed at pH 5 reduces in intensity. At pH 8, the two sets of EPR signals have comparable intensities, and at pH 9, the high field signals dominate. The variation in peak intensity with pH is shown as an inset in Fig. 1. It is clear that at pH 7.4, a mixture of two complexes is observed. Peisach and Blumberg (34Peisach J. Blumberg W.E. Arch. Biochem. Biophys. 1974; 165: 691-708Google Scholar) have shown that a combination of AII and gII values can indicate ligand type. The AII and gII values at pH 5 are most typical of three nitrogen and one oxygen ligands (3N1O), although 2N and 2O coordination cannot be ruled out. At pH 10, the AII and gII values are more typical for 4N coordination. We have carried out complementary studies using CD spectroscopy. Fig. 2 shows visible CD spectra for Aβ-(1–28) with one equivalent Cu2+ at various pH values between 4.5 and 10.5. At pH 7.5, a visible absorption band is observed at 600 nm (ϵ600 nm = ∼50 m–1 cm1), which is typical of a type II Cu2+ d-d transition. At pH 7.5 and below, the associated CD band is extremely weak and is not detected; however, the accompanying CD band at 312 nm assigned as an amide-to-copper charge transfer band is observed at pH 5.5 and above (35Fawcett T.G. Bernarducci E.E. Krogh-Jesperen K. Schugar H.J. J. Am. Chem. Soc. 1980; 102: 2598-2604Google Scholar). Only as the pH is raised to above pH 8.5 is a negative CD band at 514 nm observed, arising from Cu2+ d-d transitions. The insets in Fig. 2 show the change in the intensity of CD bands in the presence of 1 eq of Cu2+ at 252, 312, and 514 nm. Relatively strong CD bands are often observed for d-d transitions of Cu2+ tetragonal complexes (36Viles 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: 2042-2047Google Scholar, 37Garnett A.P. Viles J.H. J. Biol. Chem. 2003; 278: 6795-6802Google Scholar). However, these complexes involve main-chain amide coordination as well as histidine coordination via the imidazole ring. In these cases, the dominant contribution to optical activity observed is due to the vicinal contributions resulting when the asymmetric α-carbon is held in a chelate ring between two chelating donor atoms (e.g. adjacent main-chain amides) (38Tsangaris J.M. Martin R.B. J. Am. Chem. Soc. 1970; 92: 4255-4260Google Scholar). At physiological pH and below, the lack of optical activity from the d-d transition of the Cu-Aβ complex suggests that backbone amide coordination is not taking place. It is likely that raising the pH above 8 promotes amide deprotonation and copper coordination by the main chain, resulting in a CD band being observed at 514 nm. In summary, it is clear that Aβ forms a Type II square-planar coordination geometry with Cu2+, and both EPR and CD measurements indicate that the coordinating ligands are highly pH-dependent, a mixed species is present at physiological pH, and main-chain amide coordination is not present at lower pH values. Stoichiometry of Cu2+ Binding—EPR spectra of Aβ-(1–28) with increasing mol eq of Cu2+ have been collected in order to identify the number of copper ions binding to Aβ and are shown in Fig. 3. With increasing amounts of Cu2+ up to 1 eq, identical line shapes are observed with a commensurate increase in intensity. However, above 1 eq of copper, there is a clear shift to low field for the g⊥ signal; 2.06 at 1 eq of Cu2+ and 2.07 at 2 eq of Cu2+. The signal increases linearly in intensity up to two equivalents of copper. Comparison of the EPR signal intensities with a Cu(Gly)2 standard sample confirms that all of the EPR signals for the Cu-Aβ complex are observed and therefore rules out the possibility of EPR silent spin-coupled Cu2+. The addition of a further mol eq of copper (and up to 5 mol eq) results in no further increase in the intensity of the EPR spectra. Cu2+ ions in water will give a largely EPR silent signal at pH >7 (39Aronoff-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. Biochemistry. 2000; 39: 13760-13771Google Scholar). We have confirmed this observation by obtaining EPR spectra of CuCl2 in N-ethylmorpholine buffer at pH 7.8; copper EPR signals are drastically attenuated relative to Cu2+ bound to Aβ. Spin integration of the EPR spectra has been plotted versus copper addition as shown as an inset in Fig. 3. It is clear from the EPR data that Aβ-(1–28) binds two Cu2+ ions sequentially. After 2 mol eq of Cu2+, Aβ-(1–28) becomes saturated with copper at physiological pH. Similar studies have been performed using CD, both in the UV and visible regions. Monitoring of the band at 205 nm reveals saturation of Aβ-(1–16) and Aβ-(1–28) at 2 mol eq of Cu2+ at pH 9. In summary, both EPR- and CD-derived Cu2+ binding curves indicate Cu2+ saturation of binding sites on Aβ-(1–28) after 2 mol eq. Folding of the Backbone in the Presence of Cu2+—It is believed that amyloid formation in AD and other amyloidogenic diseases such as prion disease are the result of protein or peptide misfolding. The Aβ peptide has a structural transition associated with amyloid formation, with conversion from random coil to an extended β sheet-like conformation (40Serpell L.C. Biochim. Biophys. Acta. 2000; 1502: 16-30Google Scholar). CD spectra in the UV region can be used to monitor changes in the main-chain conformation. Fig. 4 shows the CD spectra of Aβ-(1–16) and Aβ-(1–28) with the addition of increasing amounts of Cu2+ at various pH values. Cu2+ binding to Aβ-(1–16) occurs as low at pH 5.9, since the negative signal at 198 nm is reduced in intensity as the pH is increased from pH 5.5 to 5.9 in the presence of Cu2+. Control experiments with Aβ-(1–16) in the absence of Cu2+ shows that there are no significant differences in the spectra between pH 5.2 and 9.5 (data not shown). At pH 7.5, the addition of Cu2+ (Fig. 4a) causes a loss of the negative CD band at 198 nm and the appearance of a positive band at 225 nm. The intensity of a second positive contribution at 205 nm is pH-sensitive and is more apparent at higher pH values. (For comparison, see Fig. 4, a and b, which show copper titrations at pH 7.5 and 9.5, respectively.) Similar changes in the CD spectra of Aβ-(1–28) with Cu2+ addition are observed as shown in Fig. 4c. A positive band at 225 nm appears and is accompanied by a loss of negative band at 198 nm. The positive contribution at 205 nm with copper is observed but is swamped by the more intense random coil CD band at 200 nm. Apo-subtracted difference spectra, which illustrate the effect of the Cu2+ addition to both Aβ-(1–16) and Aβ-(1–28) are shown as insets in Fig. 4, a–c. The difference spectra emphasize the similarity in the changes in the secondary structure with copper addition between Aβ-(1–16) and Aβ-(1–28). The additional 12 residues to the C terminus have no effect on the copper-induced conformational transition. The changes in the spectra with the Cu2+ addition are complete by 2 mol eq of Cu2+, which agrees with the stoichiometries determined by EPR (Fig. 3). Isodichroic points are observed at 217 and 195 nm, and these are maintained between 0 and 1.0 eq of Cu2+. If a dimeric species were formed, the isodichroic point would be expected to shift at 0.5 eq of Cu2+. The chirality at 217 nm is largely invariant with the addition of copper between pH values of 5.5 and 9.5. An increased negative CD signal at 217 nm is often attributed to β-sheet or extended conformation. The copper-induced structuring of Aβ can therefore not be directly attributed to an increase in β-sheet or extended structure. The appearance of a positive CD contribution at 205 nm is not characteristic of any defined secondary structure but does indicate increased ordering of the main chain. Affinity of Copper Binding—Key to the physiological significance of Cu2+ binding to Aβ is its affinity. With this in mind, we have used the competitive effects of glycine and l-histidine to measure Cu2+ affinity for Aβ by fluorescence spectroscopy. Fig. 5a shows that the addition of Cu2+ to Aβ-(1–28) causes marked quenching of the tyrosine fluorescence signal at 307 nm. As glycine is added, it competes with Aβ for the Cu2+, and the tyrosine fluorescence signal reappears, as shown in Fig. 5b. Cu2+ coordinates to glycine via the amino and carboxylate groups with an apparent (pH-adjusted) Ka = 1.8 *106m–1, and two glycine residues will bind to a single Cu2+ ion (33Dawson R.M.C. Elliot D.C. Elliot W.H. Jones K.M. Data for Biochemical Research. Claredon Press, Oxford, UK1986Google Scholar). It takes more than 100 mol eq of glycine to cause the tyrosine fluorescence signal to completely return to its maximal strength. Half of the maximal quenching is achieved at ∼18 ± 2 eq of glycine. Thus, the affinity of Cu2+ for Aβ-(1–28) is at least an order of magnitude higher than that of glycine, putting the dissociation constant Kd in the submicromolar range (Kd « 0.5 μm). Similar experiments have been carried out using l-histidine as the competing ligand. In this case, tyrosine fluorescence returns to its maximal value with only 2.5 mol eq of l-histidine, as shown in Fig. 5c. Two molecules of histidine will bind a single Cu2+ ion using the amino and imidazole nitrogens as ligands with an apparent Kd at pH 7.8 of 1.5 nm. This indicates that copper will bind to Aβ-(1–28) with a lower affinity than l-histidine. This puts the affinity of Cu2+ for Aβ greater than 1.8 × 106m–1 but less than 6.7 × 108m–1 or a Kd of «500 nm but >1.5 nm (i.e. 10–100 nm). We have obtained similar Cu2+ affinities for Aβ-(1–28) using CD spectroscopy. CD was used to directly measure the copper-Aβ-associated absorption band at ∼314 nm. Using the competitive effects of glycine, copper absorption bands become CD-silent when bound to nonchiral glycine (37Garnett A.P. Viles J.H. J. Biol. Chem. 2003; 278: 6795-6802Google Scholar). We find using the CD band at ∼314 nm that similar amounts of glycine are required to remove Cu2+ from Aβ-(1–28) as is indicated by the fluorescence quenching experiments. Very high affinities (1015m–1) have previously been suggested for Cu-(Aβ)2 (24Atwood C.S. Scarpa R.C. Huang X. Moir R.D. Jones W.D. Fairlie D.P. Tanzi R.E. Bush A.I. J. Neurochem. 2000; 75: 1219-1233Google Scholar) (i.e. at 0.5 eq of Cu2+). We note that glycine or l-histidine would have little impact on the binding of copper to such a high affinity site. This could result in a false plateau for the fluorescence quenching data shown in Fig. 5. To rule out this possibility, we have added substoichiometric levels of Cu2+ to Aβ (0.3 mol eq of Cu2+). If there is indeed a very high affinity site for copper associated with Aβ-(1–28), then the addition of l-histidine would have little effect on the fluorescence signal. However, the addition of just 1 eq l-histidine to Aβ-(1–28) with 0.3 mol eq of Cu2+ present caused the fluorescence signal to return to its maximal value. We can also rule out apo-Aβ being inadvertently loaded with Cu2+ during peptide synthesis and sample preparation, since no Cu2+-associated signals are observed for apo-Aβ-(1–28) in EPR or CD spectra. In summary, using both direct measurements of Cu-Aβ-(1–28) from CD absorption bands and indirect fluorescence quenching methods, we have shown that the first molar equivalent of Cu2+ ions bind to Aβ with a dissociation constant in the submicromolar level, 10–100 nm. The possibility of a high affinity copper site for Aβ-(1–28) has been ruled out. Cu2+ Coordination Ligands—Fragments of Aβ have been used to determine which residues are involved in binding to Cu2+. The three histidine residues within Aβ-(1–42) are thought to be the most likely c
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