High-resolution NMR studies of the zinc-binding site of the Alzheimer’s amyloid b-peptide Jens Danielsson1, Roberta Pierattelli2, Lucia Banci2 and Astrid Graslund1 ă Department of Biochemistry and Biophysics, Stockholm University, Sweden ` Department of Chemistry and Magnetic Resonance Center, Universita di Firenze, Sesto Fiorentino, Italy Keywords aggregation; Alzheimer amyloid b-peptide; copper binding; NMR; zinc binding Correspondence A Graslund, Department of Biochemistry ă and Biophysics, Stockholm University, S-106 91 Stockholm, Sweden Fax: +46 155597 Tel: +46 162450 E-mail: astrid@dbb.su.se (Received 22 June 2006, revised 20 October 2006, accepted 27 October 2006) doi:10.1111/j.1742-4658.2006.05563.x Metal binding to the amyloid b-peptide is suggested to be involved in the pathogenesis of Alzheimer’s disease We used high-resolution NMR to study zinc binding to amyloid b-peptide 1–40 at physiologic pH Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo-form and the holo-form, with respect to the NMR timescale This causes loss of NMR signals in the resonances affected by the binding Heteronuclear correlation experiments, 15 N-relaxation and amide proton exchange experiments on amyloid b-peptide 1–40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N-terminus, similar to a previously proposed copperbinding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem 279, 18169–18177] Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid b-peptide The dissociation constant Kd of zinc for the fragment amyloid b-peptide 1–28 was measured by fluorescence, using competitive binding studies, and that for amyloid b-peptide 1–40 was measured by NMR Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K Zinc also has a second, weaker binding site involving residues between 23 and 28 At high metal ion concentrations, the metal-induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides At low metal ion concentrations, on the other hand, the metal-induced structure of the peptide counteracts aggregation The amyloid b-peptide (Ab) is the major component of senile plaques and soluble oligomers, amyloid b-derived diffusible ligands, which are considered to play an important role in Alzheimer’s disease (AD) pathology Ab is the product of cleavage of amyloid precursor protein (APP) [1] at position(s) 39–42, which creates a soluble monomer There is evidence that APP is involved in copper homeostasis APP has a selective metal-binding site and is able to reduce bound Cu(II) to Cu(I) It also participates in the regulation of copper levels, and its expression is affected by copper concentration Also other metal ions, such as Zn2+ and Fe2+, are known to interact with APP [2–6] The Ab peptide mainly appears as a random coil in aqueous solution, but contains some secondary structure elements: A poly(proline II) helix (PII) in the N-terminus, and two b-strands in the central part and in the C-terminus [7–9] The soluble monomeric peptide has a high tendency to form Ab oligomers, which eventually produce Ab fibrils Although the monomeric peptide is not neurotoxic and neither are the fibrils, Ab oligomers have been shown to induce cognitive loss due to neurodegeneration, via Abbreviations Ab, amyloid b-peptide; AD, Alzheimer’s disease; APP, Alzheimer precursor protein; HSQC, heteronuclear single quantum coherence; LMCT, ligand to metal charge transfer 46 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS J Danielsson et al pathophysiologic mechanisms that are not completely understood The Ab oligomers are thus more neurotoxic than the Ab fibrils [10–15] The detailed structure of these Ab oligomers has yet to be resolved, although they seem to be similar to other amyloidforming peptide oligomers [16] The aggregation process is accompanied by a significant change in structure, whereby monomeric, mostly unstructured Ab folds to form oligomeric b-sheet-rich forms The detailed mechanism of this transition is not known Increased metal concentrations (mainly copper, iron and zinc) have been found in the brains of AD patients, both in the amyloid plaques (copper and zinc) and in the cortical tissue (zinc) [17,18] Interactions of copper and zinc with Ab induce peptide aggregation if the metal concentration is high enough, i.e > : metal ⁄ peptide ratio [19–21] The formed aggregate is suggested to be amorphous, and thus high concentrations of copper and zinc prevent fibril formation by promoting the formation of nonfibril aggregates [20,22–24] The N-terminal fragment Ab(1–28) has, however, been shown to undergo increased fibril formation upon interaction with zinc [25] Another N-terminal fragment of Ab, Ab(1–16), forms stable oligomers in the presence of both copper and zinc [26] On the other hand, binding of low concentrations of copper or zinc ions to the full-length Ab(1–40) at less than : metal ⁄ peptide ratio reduces the Ab oligomeric stability and prevents aggregation, whether amorphous or fibril forming [27,28] The effect of metals on Ab is thus dependent on the experimental conditions, such as pH, salt concentration and, most important, metal concentrations [21,29] In addition, Ab-bound Cu(II) may be reduced to Cu(I), and the complex may produce hydrogen peroxide, which has been suggested to be neurotoxic in AD [30] Soluble monomeric Ab has a high-affinity copperbinding site in the N-terminus, and the metal ion is suggested to coordinate in a planar configuration with the three histidines (6, 13 and 14) and the N-terminus or Tyr10 N-terminal deletions alter the binding affinity, suggesting that the N-terminal amide participates in copper coordination and that Tyr10 does not [31– 34] The metal-binding site, involving the three histidines, is also able to bind a zinc ion The fourth ligand for zinc binding has been suggested to be either Tyr10, Glu11, Arg5 or the N-terminus [35–37] Both copper and zinc have been suggested to have a second binding site The ligands of this hypothetical weaker binding site are unknown [31,38,39] The binding affinity of the metal ions to Ab is pH-dependent: copper has a higher affinity under mild Zinc-binding site of the amyloid b-peptide acidic conditions, whereas the affinity for zinc is less pH-dependent over a range of pH values between 6.5 and 7.5 [29] At pH below 6, when the histidines are mainly protonated, no zinc binding occurs [21] Under physiologic conditions, Ab has a higher propensity to bind zinc, whereas under mildly acidic conditions, as in physiologic acidosis following an inflammatory process, copper is preferentially bound [40] This difference between copper and zinc affinity at different pH values has been suggested to be a pathogenic mechanism of Ab: under normal conditions, zinc protects against copper-induced Ab toxicity, which is induced by physiologic acidosis [22,29,40] The present study shows for the first time the detailed molecular effects on the full-length Ab of zinc binding Thanks to the combined use of heteronuclear NMR, fluorescence and CD spectroscopy, a molecular model for the zinc interaction in solution is obtained We show that the high-affinity zinc-binding site is formed by three histidines and the N-terminus, whereas a second, low-affinity binding site comprises residues 23–28 The results of these studies are discussed within the context of the general mechanism of the onset of pathologic states upon Ab aggregation Results NMR spectroscopy Interaction with zinc ) heterocorrelation experiments Ab(1–40) in aqueous solution has been extensively characterized by NMR spectroscopy, mainly through two-dimensional 1H–1H NMR and 1H–15N-heteronuclear single quantum correlation (HSQC) experiments In 1H–15N correlation experiments, some signals are affected by an exchange process with the solvent and are missing in the spectra Thus, despite the good resolution, not all resonances are visible in the 1H–15N HSQC spectra In contrast, 13C-bound protons are not affected by an exchange process and show a suitable signal dispersion, which allows high-resolution characterization Therefore, the combination of these two sets of data (1H–15N and 1H–13C) provides a detailed characterization of metal binding to Ab As the metal ions could be sequestered from solution by complexation with the buffer, we can only obtain an estimate of the order of magnitude of the dissociation constants On the other hand, from NMR we obtain an unambiguous description of the binding When zinc ions are added to a solution of Ab(1–40), all the signals in 1H–15N and 1H–13C HSQC experiments are reduced in intensity At one equimolar zinc concentration, the remaining intensity fraction for the FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 47 Zinc-binding site of the amyloid b-peptide J Danielsson et al CHa signals of the residues in the C-terminal region is 0.69 ± 0.04, whereas His13 has a relative intensity of 0.15 ± 0.02, and His6 and His14 have relative intensities of 0.3 ± 0.01 This observation confirms the involvement of the histidines as metal ligands Asp1 shows a 0.60 residual signal intensity, whereas Val12 has a residual signal intensity of 0.52 ± 0.06 The signal reduction of Asp1 is equal to the reduction seen for other residues located in the N-terminal region, such as Ser8 and Arg5 Asp1, His6, Val12, His13 and His14 also show small but significant chemical shift changes (0.010–0.015 p.p.m in the proton dimension) upon zinc binding (Fig 1) The chemical shift varia- tions are probably the result of conformational changes of the backbone upon metal binding For other residues, no chemical shift changes are observed The Cb region shows the same signal intensity reduction pattern but with no or very small induced chemical shift changes The selective spectral changes observed upon zinc addition for Asp1, His6, His13 and His14 clearly indicate the involvement of these residues in metal coordination In particular, the specific changes in the Asp1 chemical shift provide a direct indication of the involvement of the N-terminus in zinc binding The chemical shift change observed for Val12 is probably due to its proximity to the histidine ligands D1 H13 H6/H14 Y10 V12 Fig 1H–13C HSQC spectra of the Ca region of 50 lM Ab(1–40) in 10 mM phosphate buffer at pH 7.4 and 286 K without (black) and with (red) 30 lM zinc as chloride All resonance peaks show reduced intensity, but some specific reduction is present for the histidines, Asp1, Tyr10 and Val12 The resonances of His6, His13, His14, Asp1 and Val12 exhibit induced chemical shift changes upon zinc binding The inset shows the broadening and chemical shift changes of Asp1 upon zinc titration with 0, 20 and 50 lM zinc, indicating the induced chemical shift changes 48 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS J Danielsson et al Zinc-binding site of the amyloid b-peptide The aromatic region of the 1H–13C HSQC spectrum is particularly informative about the effects of Zn2+ (Fig 2) The Tyr10 aromatic resonances are unchanged upon zinc addition, indicating that the aromatic ring of Tyr10 is not involved in binding of the metal Instead, the e and d resonances of the histidines show significant intensity losses, of > 80% The d-proton signals of His6 and His13 show some induced chemical shift changes, whereas His14 only exhibits intensity loss for the d-proton signal (Fig 2A) Furthermore, there is a total loss of the e-protons of the His6 and His14 signals and a weakening (> 80% reduction) of His13 resonance (Fig 2B) The zinc binding can thus be envisaged as coordination of the metal ion by the nitrogens of the imidazole rings of the histidines and the amino group in the N-terminus Use of the signal intensity reduction of the 1H–13C HSQC crosspeaks to estimate the dissociation constant of zinc yields Kd  lm at pH 7.2, in phosphate buffer, at 286 K Also, the 1H–15N HSQC spectrum shows that the reduction in intensity does not affect all the signals to the same extent This signal reduction, where the signal is lost due to addition of zinc, could as a first approximation be interpreted in terms of a first-order binding process An apparent dissociation constant appKd* can be calculated; the star indicates that the dissociation A Y10 Y10 B Fig 1H–13C HSQC spectra of the aromatic regions of 50 lM Ab(1–40) in 10 mM phosphate buffer at pH 7.4 and 286 K without (black) and with (red) 30 lM zinc as chloride (red) The d and e resonances of the histidines shown in (A) and (B), respectively, show significant signal intensity losses All three histidines are affected In (A), it is shown that H6 and H13 show some induced shift but H14 only exhibits signal intensity loss for the d-protons The e-protons shown in (B) exhibit a total loss of H6 and H14 resonances, and a weak H13 resonance remains at the same chemical shift The tyrosine aromatic crosspeaks are unaffected upon zinc addition B A H13 H6 FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS H6 H13 H14 H14 49 Zinc-binding site of the amyloid b-peptide J Danielsson et al constant calculated here is not a quantitative measure of binding of zinc by the molecule, but rather a measure of which residues are most involved in the binding Such an apparent dissociation constant was calculated for all individual resonances (Fig 3A) The apparent dissociation constant varies along the peptide chain, indicating that this is a generalized description of binding, which in turn reflects the induced changes in the peptide upon binding The C-terminus residues 29–40, which are not directly affected by the zinc interaction, show a fairly constant apparent dissociation constant, but four signals, originating from residues 23, 24, 26 and 29, are significantly more affected In the N-terminus, the regions corresponding to the binding site show a very high apparent affinity constant With a simpler approach, the decrease in intensity of the crosspeaks can be studied, and using this approach a similar pattern is observed (data not shown) It can be seen that, in addition to a reduction over the entire polypeptide sequence, a more dramatic effect is observed for the first 15 residues This behavior is similar to that observed with 13C HSQC, and can be interpreted in the same terms as specific binding of the metal in the N-terminal part of the peptide These observations confirm that the zinc-binding site is in the N-terminus, and are consistent with three histidines participating in the binding, possibly with the formation of a bend or a turn in region 7–12 and one 10 app Kd /µM A ** * ** ** * ** R2 Difference B -4 -8 Amide Proton Stability Change C 0.4 0.2 ** * ** ** * ** Relative Signal Intensity D 50 0.4 0.2 10 20 residue 30 40 Fig (A) 1H–15N HSQC signal intensity of 50 lM Ab(1–40) in 10 mM phosphate buffer (pH 7.2) at 286 K The calculated apparent dissociation constant, appKd*, of zinc for Ab A second interaction site is suggested in residues 23, 24, 26 and 28 The residues indicated with an asterisk (*) were not observed, due to rapid exchange with solvent water (B) Change in transverse relaxation rate, R2, upon addition of zinc under the same conditions as in (A) The increase in R2 upon addition of zinc (negative difference) suggests greater rigidity at the binding site (C) The amide proton exchange variation with temperature measured as described in the text The amide proton stability is increased along the whole peptide However, the stability increase is more prominent close to the binding site This approach shows a good correlation with relaxation data (D) Signal intensity decrease upon copper binding The fractional remaining intensity after addition of 20 lM copper to 50 lM Ab Copper binding shows the same pattern as zinc binding FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS J Danielsson et al Zinc-binding site of the amyloid b-peptide in region 2–5 The 1H–13C HSQC and 1H–15N HSQC results show that Tyr10 does not participate in zinc binding, supporting previous proposals for copper binding [25,31,32] The presence of a second binding site for zinc is suggested by the selective effect for some residues in the C-terminal region The general signal intensity reduction in the 1H–15N HSQC spectra due to zinc binding could have multiple origins, such as aggregation, the presence of chemical exchange rate between conformations in an intermediate regime with respect to the NMR timescale, increased amide proton exchange rate in the binding region, and ⁄ or increased relaxation rates due to decreased dynamics in the binding region We favor aggregation as the main explanation for the general intensity loss, as this should give signal reduction due to the line-broadening of the large aggregates In the 1H–13C HSQC spectrum, there is some crosspeak intensity left at high zinc concentrations, whereas there is no residual crosspeak intensity in the amide proton region of the 1H–15N HSQC spectra This different feature suggests that in the 1H–15N HSQC case, there are further mechanisms (in addition to aggregation and metal-binding effects) that lead to specific reductions in signal intensity, e.g relaxation effects and proton exchange effects In order to vary the chemical exchange rate and to be able to detect additional resonances, a 1H–15N HSQC spectrum was recorded at a lower temperature (278 K) As shown in Fig 4, four new crosspeaks appeared, suggesting that some additional residues exhibit slower exchange at lower temperatures The intensity of the new peaks was too low to attempt the assignment of the newly identified resonances Relaxation and diffusion measurements The local mobility changes upon zinc binding to Ab were studied by NMR relaxation measurements Amide HN R1 and R2 were measured The average R1 value for the peptide with no zinc bound is 2.16 ± 0.40 s)1 and is unchanged upon zinc binding This suggests that the peptide remains monomeric upon zinc binding This was also confirmed using pulsed field gradient-NMR diffusion studies Ab(1–40)’s diffusion coefficient is 1.04 ± 0.02 · 10)10 m2Ỉs)1 at 286 K, which is in fairly good agreement with the expected value of 1.09 · 10)10 m2Ỉs)1, with the viscosity changes accounted for by an empirical function [8,41] Upon zinc titration, no significant change of diffusion coefficient was measured (data not shown) All changes were within the range of experimental error, suggesting that no stable small soluble oligomers of the peptide were induced by zinc binding The zincinduced peptide aggregates immediately grow to sizes where the linewidth in the NMR spectra broadens beyond detectable limits, so the relaxation and diffusion properties of the aggregate cannot be measured with NMR G38 G29 G9 G25 G33 G37 S26 S8 E3 E22 V39 I31 V24 N27 D23 Q15 K28 D7 F4 V36 M35 F20 E11 K16 L17 Fig 1H–15N HSQC spectra of 75 lM Ab(1–40) in 10 mM phosphate buffer at pH 7.4 (278 K) and 50 lM ZnCl2 The assignment is shown Four new crosspeaks appear when the temperature is lowered and the exchange rate is therefore slowed The new crosspeaks are highlighted in circles FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS F19 Y10 V12 V18 L34 R5 A30 I32 A21 V40 51 Zinc-binding site of the amyloid b-peptide J Danielsson et al The mean R2 value of the peptide changes upon zinc addition from 4.85 ± 1.40 s)1 to 7.12 ± 6.44 s)1 Figure 3B shows the changes in R2 upon zinc binding for all the residues The C-terminal residues show no or small changes in R2, whereas the residues close to the binding site show increased R2 upon zinc binding, as expected because of the presence of a zinc-induced structure in this region To confirm that the R2 changes are due to decreased local mobility, amide proton exchange stability was measured As shown in Fig 3C, the amide protons show slightly increased stability, which is most prominent close to the binding site We conclude that zinc binding induces increased order in the N-terminus of the peptide This is reflected by the increased R2 in this region, and also by the increased amide proton stability, which is most likely due to increased protection of the amide protons from the solvent water Interaction with copper To obtain some complementary information, the interaction of copper was investigated using 1H–15N HSQC experiments and the paramagnetic broadening effect expected from bound copper Figure 3D shows that copper addition produced a significant decrease in the crosspeak intensities for residues 3, 5, 8, 9, 10, 11, 12, 16 and 17, and a total loss of the Phe4 signal when 40% of the peptide is bound to copper The signals of residues and 15 also show a signal intensity decrease, but not as prominent as for the others Crosspeaks for residues 1, 6, 13 and 14 are not visible even with no copper added As well as the selective effects described above, there is a general copper-induced reduction of the crosspeak intensities, which could be ascribed to aggregation of the peptide At high copper concentrations, the crosspeak signal of Ala21 is also lost This confirms the presence of a second, weaker binding site in the central part of the peptide All the signals affected by the addition of copper reappeared, even if not completely, upon EDTA addition (EDTA concentration exceeded metal concentration about 20-fold) (data not shown) Fluorescence spectroscopy The peptide has a tyrosine residue in the N-terminal part of the peptide, Tyr10, and the copper-binding site is close to this site, as shown above Addition of copper quenches the tyrosine fluorescence signal [31,38] Fluorescence quenching can be used to directly measure the dissociation constant of copper ions, and indirectly to estimate the dissociation constant of zinc 52 Table Dissociation constants of copper and zinc for Ab The dissociation constants were calculated from tyrosine fluorescence pH Kd (Cu2+) (lM) Kd (Zn2+) (lM) 7.2 0.4 ± 0.1a 6.5 7.2 1.2 ± 0.08a 2.5 ± 0.2c 1.1 1.2 3.2 6.6 ± ± ± ± 0.08a 0.03b 0.1a 0.2c a Dissociation constant for Ab(1–28) calculated using fluorescence; 10 mM sodium phosphate buffer, 20 °C b Dissociation constant for Ab(1–40) calculated using 1H–13C HSQC crosspeak intensity; 10 mM sodium phosphate buffer, 20 °C c Dissociation constant for Ab(1–28) calculated using fluorescence; 10 mM Hepes buffer, 20 °C Full-length Ab has a tendency to adhere to the wall of the quartz cuvette, and this slow process imparts a time dependence to the fluorescence spectra Treatment with ethylenimine [42] increased the stability, but adhesion was still detectable Use of the full-length peptide yielded an approximate affinity for copper, Kd  0.5 lm, but with large deviations of the data points from the fitted line (data not shown) The shorter fragment Ab(1–28) showed less tendency to adhere, and the sample was stable for more than days The fluorescence binding studies were therefore performed using the shorter fragment The dissociation constant for copper was determined as Kd ¼ 0.36 ± 0.1 lm for this fragment at pH 7.2, assuming a : stoichiometry (Table 1) This Kd is in good agreement with the preliminary results obtained using the full-length peptide and the dissociation constant determined earlier [31], but it is significantly higher than that earlier reported for the full-length peptide at physiologic pH [43] There is a fractional fluorescence signal left (approximately 50%) at the end of titration, suggesting that the quenching copper ions are not in direct contact with the fluorescent side chain of tyrosine, but close enough to cause partial quenching There are some systematic deviations of the residuals from the fit, probably arising from induced aggregation Including a term accounting for the induced aggregation in the binding equation essentially removes all systematic residuals (this modified model is described in supplementary Doc S1) The dissociation constant is unchanged upon extending the model with this term Zinc has no fluorescence-quenching abilities, but its binding affinity can be estimated by competitive titrations with copper, provided that the binding site is the same Ab(1–28) was incubated with various amounts of zinc Copper was added to these mixtures, and produced signal quenching similar to what was observed after the addition of copper alone (Fig 5) FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS J Danielsson et al Zinc-binding site of the amyloid b-peptide copper compete for the same binding site, the dissociation constant for zinc can be calculated using an expression for the bound fraction as a function of the dissociation constants of the two metal ions The dissociation constant for zinc was estimated to be 1.08 ± 0.08 lm, indicating only slightly lower affinity than for copper at pH 7.2, close to physiologic conditions (Table 1) This dissociation constant for zinc is in excellent agreement with that obtained from NMR data, which was measured on the full-length peptide and is also included in Table Thus, the affinities of zinc for the shorter fragment Ab(1–28) and for Ab(1– 40) are the same Under mildly acidic conditions (pH 6.5) and in phosphate buffer, the dissociation of copper is slightly increased (Kd ¼ 1.16 ± 0.08 lm), whereas the dissociation constant for zinc increases from 1.08 lm to 3.19 ± 0.08 lm (Table 1) The changes in affinity due to changes in pH close to pH are not large To investigate the effect of the buffer used in these experiments, the fluorescence measurements were repeated in 10 and 50 mm Hepes buffer at pH 7.2 and with 0, 10 and 20 lm Zn2+ Under these conditions, the dissociation constant for the Ab(1–28)–copper interaction was determined to be 2.5 ± 0.2 lm, and that for the Ab(1–28)–zinc interaction was 6.6 ± 0.1 lm, similar to what was estimated in the experiments using phosphate buffer Fluorescence intensity / I/I0 0.8 0.6 0.4 0.02 Residuals 0.01 CD spectroscopy -0.01 -0.02 10 20 Copper Concentration / [µM] 30 Fig The tyrosine fluorescence intensity of 10 lM Ab(1–28) in 25 mM phosphate buffer (pH 7.2) at 298 K and 305 nm as a function of copper concentration The three datasets correspond to (s), 60 (n) and 90 (h) lM zinc acetate added The solid lines are the fitted curves of the one-to-one binding equation The plateau values differ between the attenuating intensities This could be due to different peptide aggregation propensities at different total metal ionic strengths In the bottom panel, the residuals from the fit are shown Addition of EDTA to release the copper from the peptide by competition recovered most but not all of the tyrosine fluorescent signal Approximately 30% was still missing, suggesting that this fraction is aggregated and ⁄ or precipitated Assuming that zinc and To monitor the structural changes of Ab induced by metal ion interactions, CD spectra were recorded for Ab with increasing amounts of added copper and zinc (supplementary Fig S1) The interaction of Ab with copper reduces the amount of PII helix present in apo-Ab, as demonstrated by examining the difference spectra The N-terminal part of the Ab peptide has a relatively high propensity to adopt a PII helix [8] Thus, a reduction of PII helix secondary structure is consistent with a copper interaction in the N-terminal part of the peptide At low copper concentrations, the structural transition is between two states, and therefore the CD spectra show an isoelliptic point Zinc binding gives similar, but not identical, results to those obtained with copper The zinc interaction mainly reduces the signal intensity but does not clearly reduce the amount of PII helix This may be due to induced aggregation and subsequent precipitation At neutral pH, as in this present study, zinc has a higher propensity to induce aggregation than copper [40] This aggregation can mask the structural change of the peptide FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 53 Zinc-binding site of the amyloid b-peptide J Danielsson et al Discussion Zinc is suggested to have a major effect on aggregation of Ab [19–21,44], either increasing the aggregation at high zinc concentrations or reducing the aggregation at low concentrations [27,28] Here we have studied the zinc-binding site in soluble monomeric Ab Both zinc and copper induce specific NMR changes, affecting the same residues in the peptide This is supported by the fluorescence data, which also show that zinc and copper compete for the same high-affinity binding-site (Fig 5) This in agreement with the findings for the shorter fragments Ab(1–16) and Ab(1–28) [45] However, both copper and zinc have a putative second weaker binding site, as shown by NMR This is in agreement with the finding of two binding sites for copper in earlier studies [31,33] The details of the high-affinity binding site for zinc were studied by NMR 1H–13C HSQC and 1H–15N HSQC experiments showed a selective zinc-binding site with His6, His13 and His14 and the N-terminal Asp1 as ligands (Fig 1) Direct study of the 1H–13C HSQC crosspeaks of the aromatic amino acid side chains shows that Tyr10 is not directly involved in the binding, but is located close to the bound metal (Fig 2) The quenching effect of copper on the tyrosine fluorescence signal confirms this view From the present data, a second binding site for zinc can be proposed, which involves residues 23, 24, 26 and 28 For Cu2+, a similar central region is involved, manifested by a loss of signal intensity of residue 21 A more detailed study of the second binding site of copper is not possible, due to the general paramagnetic line-broadening exhibited by copper Different ligands for metal coordination by Ab were suggested in earlier studies, but they were mainly performed on truncated fragments of Ab with or without acetylated N-terminals All studies, however, showed the histidines to be necessary ligands [40,46,47] The truncated fragments show varying binding modes with respect to the fourth ligand Acetylation of the N-terminus does not inhibit zinc binding to the N-terminal fragment Ab(1–16), but the fourth ligand is proposed to be Glu11 in this variant [36] In the same fragment but without an acetylated N-terminus, Asp1 was suggested to be the fourth ligand [35] Recently Syme et al published a study on Ab(1–16) and Ab(1–28) in which they also suggested the fourth ligand to be the N-terminus, and indeed an N-terminal-blocked variant of Ab(1–28) showed less effects when zinc was added [45] In a recent paper by Hou et al., the interaction of copper and zinc with full-length Ab(1–40) was studied using 1H–15N HSQC They suggested that, after 54 anchoring of the copper ion by the histidine side chains, a less precise binding mode of metal prevails for full-length Ab, compared to the shorter fragments They also observed a reduction on signal intensity that they interpreted as being due either to deprotonation of amides or line-broadening due to an intermediate chemical exchange rate between the apo-form and holo-form [48] In the present study, we used the fulllength Ab(1–40) and combined the use of 1H–13C HSQC and 1H –15N HSQC Under these conditions, zinc binding occurs with His6, His13, His14 and Asp1 as ligands The binding seems to be specific and affects mainly the ligands and the neighboring residues The detected residue-specific signal loss upon metal binding arises from line-broadening due to chemical exchange between conformations in an intermediate rate regime with respect to the NMR timescale (Fig 1) The N-terminal Asp1 may bind zinc either with the amine group or with the carboxylate groups on the side chain Our data give no direct evidence for which of these is responsible for binding However, the H–13C HSQC findings shows that the Ca crosspeak of Asp1 is more affected by zinc binding (both intensity and chemical shift) than are the Cb crosspeaks (data not shown) The reason could be that Ca is closer than Cb to the binding site, suggesting zinc binding to the amine group of Asp1, in agreement with the recent findings of Mekmouche et al [35] However, our data cannot rule out the possibility of zinc binding to the Asp1 side chain, and this would be in agreement with EPR studies that have reported copper coordination by three nitrogens and one oxygen, 3N1O, suggesting involvement of the carboxylate oxygen in divalent metal binding [32,47] The dissociation constant Kd for copper and Ab has been reported earlier to be approximately 1–5 lm [29,38,48] For zinc, a Kd of 3–300 lm has been reported for full-length Ab(1–40) [38,49,50] Our present results (Table 1) show micromolar dissociation constants for both copper and zinc, with a somewhat higher affinity for copper, in good agreement with the earlier reports This holds for experiments in phosphate as well as in Hepes buffer NMR measurements were similar to fluorescence measurements made under the same conditions Use of the induced chemical shift changes to estimate the dissociation constant yields Kd  2.6 lm (supplementary Fig S2) This is close to the values obtained with other techniques, and hence provides further evidence for N-terminal involvement in the metal binding of Ab As previously mentioned, these quantitative results may be somewhat biased, due to metal–phosphate complex formation and peptide aggregation, which in turn may depend on precise FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS J Danielsson et al H N N-terminus N H N N N N N CH2 H His6 H experimental conditions such as choice of buffer and temperature Binding of zinc to Ab does not induce any such well-defined structure of the peptide that can be determined by NMR methods This is in agreement with previous reports [45,48] However, the relaxation data show that the N-terminus becomes more structured upon zinc binding The results indicate that the N-terminal region folds around the ion, similar to earlier suggested structures induced by copper [31,33,40] The CD data show that copper and zinc have only minor and somewhat different effects on the spectra The reason may be that copper binding to the histidines induces a charge transfer from the ligand imidazole to the metal (so called ligand to metal charge transfer, LMCT), and thus changes the chiral properties of the peptide The copper-induced changes may therefore have this origin, and need not necessarily be due to a change in secondary structure Zinc does not have this effect on the histidines, and the zinc-induced changes in CD are very small We conclude that CD under the present conditions does not provide much information on the potential metal-induced changes in the secondary structure of Ab From the relaxation and diffusion data, we conclude that no stable, soluble, metal-induced dimers ⁄ oligomers are present This is in contrast to what has been reported for Ab(1–16) [26] The full-length peptide differs from the shorter fragments also in this respect Our results give rise to a model of the induced structure of the peptide when bound to zinc (Fig 6) The binding involves the histidines and the N-terminus There is a turn at Glu3, bending the N-terminus towards His6 We propose a second turn at Gly9, to put His13 and His14 close to the metal ion The model is similar to the model of Ab(1–28) bound to copper, proposed by Syme et al [31] We also propose that zinc has a second, possibly cooperative, binding site involving the middle segment Asp23, Val24, Asn26 and Lys28 with an induced turn at Gly25 The N-terminal region of free Ab in aqueous solution has an extended conformation rich in PII helix that is proposed to help to keep the peptide soluble and protected from amorphous aggregation [8,51–53] When the N-terminus binds zinc (or copper), it folds around the metal, forming another relatively welldefined structure The previous reports on differential metal-binding effects [20,27] on Ab aggregation at low and high metal ion concentrations may now be understood in the following terms Metal ions at high concentrations saturate the binding site(s) of Ab and lower the electrostatic repulsion between the overall negatively charged Abs (net charge nominally ) at pH 7) This effect predominates at high metal ion con- Zinc-binding site of the amyloid b-peptide CH2 CH2 His14 His13 C-terminus Fig A schematic representation of the structural model of Ab binding zinc or copper The structure was constructed using a combination of signal intensity changes, relaxation data and induced amide proton stability centrations, and explains the higher aggregation propensity under these conditions The structure induction brought about by the metal-induced fold of the N-terminus counteracts aggregation This effect, masked at high metal ion concentrations, should dominate at low ion concentrations, thereby explaining the decreased aggregation propensity under these conditions Experimental procedures The peptides, unlabeled Ab(1–40), as well as 15N-labeled and the 13C–15-N-labeled Ab(1–40), were purchased from rPeptide (Athens, GA, USA) and were used without further purification Ab(1–28) was purchased from Neosystems (Strasbourg, France) All peptides were nonmodified in the termini Solvation of the peptide was performed using the protocol suggested by Zagorski et al [54] This protocol prescribes that the peptide is dissolved in a base, here 10 mm NaOH, at high concentration (up to mgỈmL)1), and sonicated in a water ⁄ ice bath for The stock solution was diluted first with water, and then with buffer to the desired concentration and pH In the present study, the NMR peptide concentration was 50–80 lm and the pH was 7.0–7.3 The peptide was in 10 mm phosphate buffer, and NMR samples contained 10% D2O The peptide and FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 55 Zinc-binding site of the amyloid b-peptide J Danielsson et al solvents was kept cold, < °C throughout the sample preparation The pH was adjusted using NaH2PO4 and Na2HPO4 and measured using an Orion PerpHecT pHmeter (San Diego, CA, USA) The peptide concentrations of the samples were determined by weight The metal ions were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (Steinheim, Germany) as chloride and acetate The metal salt purity was higher than 98.2% The metal titrations were performed as metal ion additions to the peptide sample For each titration, a freshly prepared sample was used Small amounts of highly concentrated zinc or copper solution, as chloride or acetate, were added to the peptide The small changes in volume were taken into account in the binding constant calculations Diffusion experiments were performed using a pulsedfield gradient sequence including an eddy current delay and longitudinal storage with mono-phase square-shaped gradient pulses of 32 strengths The gradient pulse was ms and the diffusion length 100 ms Nonlinear gradient profiles were accounted for using the modified Stejskal– Tanner equation method developed by Damberg et al [56] Amide proton exchange was estimated by studying the temperature stability of the 15N HSQC peak intensity The peak intensities at 281 K (S8) and 289 K (S16) with and without zinc were compared The intensity ratio S16 ⁄ S8 is a coarse measure of the temperature stability of the amide proton, inversely proportional to the exchange propensity of the amide proton Comparing this ratio for the apo-form and holo-form of the peptide gives an estimate of the amide proton stability change upon metal interaction CD spectroscopy Acknowledgements CD spectra were measured using a Jasco (Easton, MO, USA) J-720 spectropolarimeter equipped with a PTC-343 temperature controller A mm path-length quartz cell was used, and the spectral range was 190–250 nm The resolution was 0.2 nm, and the bandwidth was nm The background was corrected for in all spectra The CD signal, in mdeg, was converted to molar ellipticity This study was supported by a grant from the Swedish Research Council and by the European Commission, Contracts LSHG-CT-2004-51 and QLK3-CT-200201989 Titrations Fluorescence Fluorescence spectra were collected using a Jobin Yvon Horiba Fluorolog (Longjumeau, France) spectrometer A mm quartz cuvette was used The excitation wavelength was 276 nm, and the measured emission was between 290 and 350 nm The excitation and emission slits were nm NMR NMR experiments were performed on Bruker (Karlsruhe, Germany) Avance 500, 800 and 900 MHz spectrometers, all equipped with cryogenically cooled probeheads A Varian (Palo Alto, CA, USA) 800 MHz spectrometer was also used All NMR experiments were performed at 286 K and pH 7.0–7.3, unless stated otherwise The 1H–15N HSQC and 1H–13C HSQC experiments were performed using 2048 · 128 increments and 32 scans, and with the carrier placed on the water resonance frequency 15N relaxation experiments were performed using the same parameters as above The 15N backbone longitudinal relaxation rates, R1, were measured as previously described [55], using delays in the pulse sequence of 20, 40, 80, 160, 320, 640 and 1280 ms, and for transverse relaxation rates, R2, the delays were 16, 32, 64, 128, 240 and 500 ms Peak intensities were fitted to a single exponential decay 56 References Hardy J & Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics Science 297, 353–356 Bellingham SA, Ciccotosto GD, Needham BE, Fodero LR, White AR, Masters CL, Cappai R & Camakaris J (2004) Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in 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148, 343–348 Supplementary material The following supplementary material is available online: Doc S1 Extended binding model A theoretical model for metal binding using tyrosine fluorescence quenching data, including a correction for metal-induced peptide aggregation Fig S1 CD spectra of 15 lm Ab(1-40) in 25 mm phosphate buffer (pH 7.2) at 298 K with increasing amounts of metal ion (A) CD spectra of Ab with 0, 3, 6, 12, 18 and 24 lm copper acetate The arrow indicates the apo-form of Ab (B) Ab(1-40) with 0, 3, 6, 9, 15 and 21 lm zinc acetate With no metal, the random coil conformation is predominant (C, D) The Zinc-binding site of the amyloid b-peptide respective difference spectra with increasing amounts of metal added to Ab (C) The difference spectrum shows the features of a PII helix, indicating that binding of copper induces a loss of PII helix The arrows indicate the change in the difference spectra with increased metal concentration Fig S2 1H NMR chemical shift of Asp1 as a function of zinc concentration The fitted line is a firstorder one-to-one binding model, and the fitting yielded Kd ¼ 2.6 lm The Ab concentration was 50 lm, in 10 mM sodium phosphate buffer at pH 7.2 and 286 K Zinc was added as chloride, and the three data points correspond to 0, 20 and 50 lm Zn2+ This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 46–59 ª 2006 The Authors Journal compilation ª 2006 FEBS 59 ... envisaged as coordination of the metal ion by the nitrogens of the imidazole rings of the histidines and the amino group in the N-terminus Use of the signal intensity reduction of the 1H–13C HSQC crosspeaks... J Danielsson et al Zinc-binding site of the amyloid b-peptide The aromatic region of the 1H–13C HSQC spectrum is particularly informative about the effects of Zn2+ (Fig 2) The Tyr10 aromatic... complexation with the buffer, we can only obtain an estimate of the order of magnitude of the dissociation constants On the other hand, from NMR we obtain an unambiguous description of the binding