Oligopeptides Generated by Neprilysin ... - ACS Publications

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Oligopeptides Generated by Neprilysin Degradation of β‑Amyloid Have the Highest Cu(II) Affinity in the Whole Aβ Family Karolina Bossak-Ahmad,†,# Mariusz Mital,†,‡,# Dawid Płonka,† Simon C. Drew,*,†,§ and Wojciech Bal*,† †

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland Florey Department of Neuroscience and Mental Health and §Department of Medicine (Royal Melbourne Hospital), The University of Melbourne, Melbourne, Victoria 3010, Australia

‡

Inorg. Chem. Downloaded from pubs.acs.org by YORK UNIV on 12/25/18. For personal use only.

S Supporting Information *

ABSTRACT: The catabolism of β-amyloid (Aβ) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aβ4−9 and a minor Aβ12−x species. Alternative processing of the amyloid precursor protein by β-secretase also generates the Aβ11−x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aβ4−9), Glu-Val-His-His-Gln-Lys-am (Aβ11−16), Val-HisHis-Gln-Lys-am (Aβ12−16), and pGlu-Val-His-His-Gln-Lysam (pAβ11−16), and investigated their Cu(II) binding properties using potentiometry, and UV−vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aβ4−9), 9.5 fM (Aβ12−16), and 1.8 pM (Aβ11−16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAβ11−16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aβ4−9 and Aβ12−16 are the highest among the Cu(II) complexes of Aβ peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aβ4−9, a major Cu(II) binding Aβ fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.

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INTRODUCTION Alzheimer’s disease (AD) is a progressive dementia involving neuronal death, cognitive decline, and deposition of plaques comprised of fibrillary β-amyloid (Aβ) peptides. According to the “amyloid cascade hypothesis”, oligomerization of Aβ due to its impaired clearance (sporadic disease) and/or overproduction (familial disease) is the initial event in the development of AD.1 Aβ is created via an amyloidogenic pathway involving sequential cleavage of the amyloid precursor protein (APP) by β-secretase (β-site APP cleaving enzyme 1; BACE1) and γ-secretase. A non-amyloidogenic pathway has also been identified, whereby APP is cleaved by α-secretase and γ-secretase. Although most familial cases of AD can be linked to an overproduction of Aβ, mostly related to mutations in APP, the more prevalent sporadic form of AD is rather associated with reduced clearance of Aβ across the blood-brain barrier and impaired Aβ degradation by dedicated proteases.2,3 The best characterized Aβ peptides constitute species starting at Asp1 created by β-secretase (Aβ1−x, with x typically 40 or 42). However, many other forms with truncated Ntermini have been identified. Early analyses of the amyloid plaque core extracted from the post mortem brain of AD © XXXX American Chemical Society

subjects identified a significant proportion of Aβ peptides with ragged N-termini, especially those truncated at Phe4.4−7 The relevance of N-terminally truncated Aβ recently regained attention, with mass spectrometry confirming the relative abundance of Aβ4−42 in both the AD brain and in unaffected individuals;8−10 while hippocampal Aβ4−42 is clearly elevated in AD relative to control subjects, it is noteworthy that cortical Aβ4−42 levels were reported to be comparable in both groups, in addition to being similar to Aβ1−42 levels.9 Another commonly reported N-truncated species begins at Glu11, which results from an alternate β′ cleavage of APP by βsecretase11 and can exist with both a free (Aβ11−x) and cyclized amino terminus (pyroglutamate Aβ11−x).6,12 A rarer Ntruncation begins at Val12 (Aβ12−x), which was reported together with Aβ4−x and other N-truncated species in early isolates of neurofibrillary tangles but not in extracellular amyloid plaque cores.13 Most N-terminal truncations are created by various enzymes taking part in the normal process of Aβ degradation. Many ZnReceived: October 29, 2018

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DOI: 10.1021/acs.inorgchem.8b03051 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry dependent proteases and peptidases exhibit an ability to cleave Aβ either in vitro or in vivo, such as neprilysin (NEP),14−17 insulin degrading enzyme (IDE),15,18 endothelin-converting enzyme,14,19 angiotensin-converting enzyme,20,21 and plasmin.22,23 NEP and IDE are believed to play an important role in the catabolism of Aβ and, through changes in their expression and activity, in the pathology of AD.24 Previous studies showed that NEP can intra- and extracellularly degrade both monomeric and oligomeric forms of Aβ1−40 and Aβ1−42.25 An inverse correlation has been established between NEP levels and activity with brain Aβ levels and AD, including a decrease of NEP in the cerebrospinal fluid of prodromal AD patients due to decreased expression.26−28 Similarly, the level of IDE was reported to be reduced in the hippocampus of AD patients.29 While there are some caveats to this correlation,30,31 the above observations are supported by murine models of AD that reported an increased level of Aβ following direct administration of the NEP inhibitor thiorphan into the hippocampus,32,33 and in response to genetic ablation of NEP34−36 or IDE.37,38 Conversely, overexpression of either NEP or IDE slowed down or reversed Aβ deposition.39−41 Cell culture studies demonstrated that IDE can degrade endogenous and synthetic Aβ, with overexpression of IDE leading to a significant reduction in levels of intracellular soluble Aβ and extracellular Aβ1−40 and Aβ1−42.42,43 Our recent study of Aβ1−40 proteolysis by NEP showed the formation of Aβ4−9 and other N-terminal fragments including Aβ10−16/17 (Figure 1). During the proteolysis of Aβ1−16, the Aβ10−16 fragment also underwent further cleavage to the final products, Aβ10−11 and Aβ12−16.17 We further demonstrated that NEP undergoes noncompetitive inhibition by Cu(II) ions.17

Similar observations were reported for IDE, which was competitively inhibited by Cu(II) and irreversibly inhibited by Cu(I) ions.44,45 A key feature of Aβ4−9, Aβ12−16, and Aβ11−16, which may be formed by NEP, IDE, and BACE1 enzymes, is the amino-terminal (H2N-Xaa-Yaa-His-) Cu(II) binding motif (ATCUN), also referred to as the N-terminal site (NTS), characterized by the His residue at the third position from the N-terminus. This site enables high affinity Cu(II) binding46−49 with Aβ4−16 binding Cu(II) with Kd = 30 fM at pH 7.4.50 Reduction of CuAβ4−16 to Cu(I) appears possible, but relies on a dynamic equilibrium with the low affinity secondary Cu(II) binding site (Kd = 0.19 μM at pH 7.4)50 involving His13 and His14,51 with electron transfer being too slow to observe a current response using conventional voltammetry.50−52 The proteolysis of CuAβ1−16 by NEP leads to a transfer of Cu(II) from Aβ1−16 to Aβ4−9 and Aβ12−16 as these fragments are formed.17 The Aβ12−x and Aβ11−x peptides contain another His residue at the second (H2N-Xaa-His-His-) or fourth (H2N-Xaa-Yaa-His-His-) position, respectively. Additionally, Aβ11−x contains a glutamic acid residue at the first position, which can spontaneously cyclize into pyroglutamic acid, a process that also occurs for the Aβ3−x peptide.53,54 We previously proposed that N-truncated Aβ4−42 may play an important role in brain copper homeostasis, serving as a metallothionein-independent synaptic Cu(II) scavenger.50,55,56 The soluble, low-molecular-weight ATCUN motifs generated by NEP from Aβ may augment or modulate this role,17 because these peptides have similar Cu(II) binding properties but are not prone to aggregation like Aβ4−42, which also has affinity for membranes.57−59 In this study, we therefore characterize in detail the Cu(II) binding properties of Aβ4−9 and Aβ12−16, as well as the Aβ11−16 and non-ATCUN pAβ11−16 peptides representative of β′ cleavage products by BACE1.

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MATERIALS AND METHODS

Chemicals. N-α-9-Fluorenylmethyloxycarbonyl (Fmoc) amino acids were purchased from Novabiochem. Trifluoroacetic acid (TFA), piperidine, and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol1-yl)uronium hexafluorophosphate (HBTU) were from Merck. Triisopropylsilane (TIS), N,N-diisopropylethylamine (DIEA), CuCl2, NaOH, HCl, KNO3, HNO3, imidazole, glycine, and pyroglutamic acid were obtained from Sigma. The TentaGel S RAM and Fmoc-Gly TentaGel S PHB resins were purchased from RAPP Polymere. Diethyl ether and dichloromethane (DCM) were from Chempur. Acetonitrile and calibrated 0.1 M NaOH solution for potentiometry were from POCH and dimethylformamide (DMF) from Roth. Peptide Synthesis and Purification. The FRHDSG-OH (Aβ 4 −9 ), FRHDSGYEV-amide (Aβ 4 −12 ), EVHHQK-amide (Aβ11−16), pEVHHQK-amide (pAβ11−16), and VHHQK-amide (Aβ12−16) peptides were synthesized using a standard Fmoc/tBu procedure and the automated microwave peptide synthesizer (Liberty1, CEM Corporation), with HBTU and DIEA as coupling reagents and 20% piperidine in DMF as a Fmoc removal agent.60 TentaGel S RAM resin was used as a solid phase, except for Aβ4−9 where Fmoc Gly TentaGel S PHB was used. Elongated peptides were cleaved from the resin in TFA/TIS/water 95:2.5:2.5 v/v/v for 2 h. Next, the peptides were precipitated from the cleavage solution with cold diethyl ether, collected by centrifugation, and the pellet was dissolved in water and lyophilized. The peptides were purified by HPLC (Waters) with UV−vis detection set at 220 and 280 nm. The eluting solvent A was 0.1% (v/v) TFA in water, and solvent B was 0.1% (v/v) TFA in 90% (v/v) acetonitrile. The concentrations of peptides were determined using the following extinction coefficients

Figure 1. Products of proteolysis of Aβ1−40 by recombinant human NEP17 and BACE. ATCUN motifs are outlined in green. No significant quantities of Aβ4−x (x > 9) are produced by NEP, since cleavage of the Gly9−Tyr10 bond is relatively fast; the Aβ4−9 fragment is created from the Aβ1−9 precursor. Red/gray dashed lines: faster/slower cleavage events. Blue dashed lines: BACE β/β′ cleavage sites of the amyloid precursor protein. Red/gray filled rectangles: major/minor cleavage products detected. * major cleavage fragments detected only during digestion of Aβ1−16-am peptide. † < 1% of NEP cleavage products. B


Article

Inorganic Chemistry at 214 nm: Aβ4−9, 15 155 M−1 cm−1; Aβ11−16 and pAβ11−16, 16 092 M−1 cm−1; Aβ12−16 15 091 M−1 cm−1.61 UV−vis and Circular Dichroism (CD) Spectroscopy. The UV−vis and CD experiments were performed at 25 °C, using either a Lambda 950 (PerkinElmer) or a BMG Labtech SPECTROstar Nano (BMG Labtech) spectrophotometer for UV−vis measurements and a J-815 CD spectrometer (JASCO). All titrations were carried out in 1 cm path length quartz cuvettes (Hellma). The pH-metric titrations were performed for 1 mM peptides acidified to pH ≈ 2. Next, CuCl2 was added to a final concentration of 0.9 mM (0.8 mM for Aβ4−9). Samples were titrated with small amounts of NaOH in a pH range 2.0−11.5. Data smoothing (10 point moving average) was applied to spectra obtained with the SPECTROstar Nano. Electron paramagnetic resonance (EPR) Spectroscopy. Firstharmonic, continuous-wave EPR (CW-EPR) spectra were obtained using a Bruker Elexsys E500 spectrometer fitted with a Bruker superhigh-Q probehead (ER 4122SHQE). Sample composition was identical to that used elsewhere except for the use of 65CuSO4 and addition of 10% v/v glycerol to prevent separation into a purified ice phase and concentrated solute phase upon freezing. The final pH of the samples was measured using a Ag/AgCl microelectrode and controlled using concentrated NaOH or HCl as appropriate, and then transferred to quartz EPR tubes and flash frozen in liquid nitrogen. Samples were contained in 4 mm OD quartz tubes inserted in a quartz coldfinger (Wilmad, WG-816-B-Q). Baseline correction of the experimental spectra was carried out by subtracting a third order polynomial fit function. Instrumental settings: microwave frequency, 9.45 GHz; microwave power, 1 mW; magnetic field modulation amplitude, 5 G; field modulation frequency, 100 kHz; receiver time constant, 41 ms; receiver gain, 47 dB; sweep rate, 13.3 gauss s−1. For each spectrum, the total integrated intensity was computed (by double-integration of the first-harmonic spectrum) and used as a normalization factor. The principal g|| and A||[65Cu] parameters characterizing each coordination mode were determined from numerical simulations of the EPR spectra using Easyspin v.5.2.15.62 A second order perturbation approximation to the spin Hamiltonian was solved using the “pepper” function in Easyspin, and the resultant value of g|| was corrected using the spectrum of the BDPA radical (g = 2.00353) as a magnetic field (B0) calibration standard. Fluorescence Spectroscopy. Fluorescence spectra for the competition experiments between Aβ4−12 and Aβ12−16 were recorded at 25 °C using an EnSpire Multimode plate reader (PerkinElmer) and a black quartz 96-well microplate (Hellma). Solutions of CuCl2 and peptide were combined to yield final concentrations of 0.20 mM Aβ, 0.18 mM Cu(II) in 20 mM HEPES, pH 7.4. Data were averaged from duplicate samples (100 μL/well) using excitation and emission wavelengths of 280 and 305 nm, respectively, and analyzed using Prism version 7.04 (Graphpad Software Inc.). The raw fluorescence intensities were converted to relative fluorescence units (RFU) by normalizing all data relative to the fluorescence intensity of Aβ4−12 at t = 0 h. Potentiometry. Potentiometric titrations of the studied peptides were performed on a 907 Titrando automatic titrator (Metrohm), using a Biotrode combined glass electrode (Metrohm), calibrated daily by nitric acid titrations.63 The 0.1 M NaOH solution (carbon dioxide free) was used as titrant. The 1.5 mL samples contained ∼1.0 mM peptides dissolved in 4 mM HNO3 and 96 mM KNO3. The Cu(II) samples were prepared by adding CuCl2 solutions to obtain 1:0.9 and 1:0.45 peptide-to-metal molar ratios. The pH range for all potentiometric experiments was from 2.4 to 11.6. All experiments were performed under argon at 25 °C. The collected data were analyzed using the SUPERQUAD and HYPERQUAD programs.64,65

basicity. The four macroscopic pKa values determined for this peptide by potentiometry (Table 1) are typical, and differences Table 1. Protonation Constants of Aβ4−9, Aβ11−16, pAβ11−16, and Aβ12−16 Peptides, Determined by Potentiometry at 25 °C and I = 0.1 M, and the Corresponding pKa valuesa peptide

species/site H5L H4L H3L H2L HL COOH (Cterm/Asp or Glu) NImH (average) NH3+ (Nterm/ Lys)

FRHDSG (Aβ4−9)

EVHHQKam (Aβ11−16)

20.07 17.47 13.63 7.37

Log β Valuesb 34.49 30.70 24.63 18.00 10.15 pKa Values −/3.79

2.60/3.84 6.26 7.37/−

pEVHHQKam (pAβ11−16)

VHHQK-am (Aβ12−16)

22.74 16.89 10.19

29.81 24.08 17.67 10.01

−/−

−/−

6.07, 5.85, 6.63 (6.35) 6.70 (6.28) 7.85/10.15 −/10.19

5.73, 6.41 (6.07) 7.66/10.01

Standard errors are less than 0.01 log units on log β values in all cases. bβ(HnL) = [HnL]/([L][H+])n. a

between them are higher than 1.1 log unit in all cases, indicating that the validity of assignments of macroscopic protonation constants to individual groups is at least 90%.66 Two Cu(II) complexes were detected by potentiometry, but only one spectroscopic species could be detected by UV−vis and CD (Figure 2A,B). This species was formed directly from the Cu2+ ion, as evidenced by the isosbestic point at 684 nm in UV−vis spectra and the presence of a single set of CD spectroscopic signals. The parameters of this species, presented in Table 2, are very typical for NTS/ATCUN motifs, involving the four nitrogen (4N) square-planar coordination.46,47,67−71 EPR spectroscopy yielded an additional species in the pH range 3−5 (Figure 2C), most likely with 1N3O coordination, that was not detected at room temperature by other methods and therefore attributed to a freezing-induced artifact (dashed red lines in Figure 2C). The global fitting of UV−vis and CD band intensities yielded uniform pKa and Hill coefficient n values (Table 1, Figure S1). These features can be easily reconciled by assuming that the CuH−1L complex differs from the CuH−2L complex by (de)protonation of the nonbonding Asp residue. The pKa for this process, 4.69, is higher from that for the free peptide by slightly less than one pH unit, which can be readily explained by a different charge of the deprotonating species: +1 for the apopeptide vs −1 for the complex. The match between potentiometric and spectroscopic results is excellent (Figure 2D), confirming the stoichiometric model and the affinity constant derived from potentiometry. Acid−Base and Complex-Forming Properties of EVHHQK-am (Aβ11−16). This peptide carries five protonating residues. Starting from the most acidic, they are the side chain carboxyl of Glu11, the imidazole side chain of His13 and His14, the N-terminal amine, and the Lys16 side chain amine, making it a H5L ligand. The protonation constants presented in Table 1 are easily assignable on the basis of the literature in all cases except for individual His residues.66 The pKa difference between the macroconstants derived from potenti-

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RESULTS Acid−Base and Complex-Forming Properties of FRHDSG (Aβ4−9). This hexapeptide contains four protonexchanging groups: the main chain (Gly9) carboxylate, the Asp7 side chain carboxylate, the His6 imidazole, and the Nterminal (Phe4) amine, listed in the order of increasing C


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Inorganic Chemistry

Figure 2. (A) UV−vis, (B) CD, (C) CW-EPR, and (D) species distribution plot of the pH-dependent Cu(II) complexes formed by Aβ4−9. Groups of vertical lines in panel C indicate the 65Cu hyperfine splitting (A||) associated with each complex (red lines correspond to freezing-induced species not identified by potentiometric titrations). The pH dependence of selected spectroscopic parameters from UV−vis (525 nm) and CD (275 nm) is overlaid with the species distribution in panel D.

Table 2. Spectroscopic Parameters for Cu(II) Complexes of Aβ4−9, Aβ11−16, pAβ11−16, and Aβ12−16 Peptides at 25 °C UV−vis stoichiometry

binding mode

λmax (nm)

CD

ε (M

−1

−1

cm )

FRHDSG (Aβ4−9) 100

CuH−1L + CuH−2L

4N

525a

CuHL + CuL + CuH−1L + CuH−2L

4N

EVHHQK-am (Aβ11−16) 520a 106

CuH2L CuHL + CuL CuH−1L + CuH−2L + CuH−3L

1N 2N 4N

CuHL CuL + CuH−1L + CuH−2L

3N 4N

λext (nm)

EPR −1

Δε (M

−1

cm )

g∥

A∥[65Cu] (10−4 cm−1)

565a 487a 311b 275c

−0.48 +0.41 +1.35 −1.65

2.184

214

570a 488a 311b 271c

−0.67 +0.32 +1.31 −2.82

2.181

218

2.363 2.275 2.182

153 187 218

2.221 2.176

204 218

pEVHHQK-am (pAβ11−16) ∼750 ∼15 650a,d 26 252e a 528 111 578a 488a 321b 270c VHHQK-am (Aβ12−16) 591a 60 517a 99 556a 480a 314b 275c

+1.72 −0.22 +1.29 −1.15 −1.75

−0.57 +0.41 +0.89 −2.89

d−d band. bN−/NIm−Cu CT band. cNH2−Cu CT band. dParameters for the CuHL complex are shown. eNIm−Cu CT band.

a

the Aβ11−16 peptide is ca. 6.35, the average of the two macroconstants. The UV−vis and CD spectra of Cu(II) complexes of Aβ11−16 (Figure 3A,B) indicate the presence of a single 4N spectroscopic species, covering all four stoichiometric forms

ometry is 0.56 log units, within the experimental error of the theoretical value of 0.6 for two totally equivalent proton releasing sites66 (noting that the pKa values are obtained by subtraction of log β values, thus their standard error is ∼0.02 pH units). Therefore, the microconstant value for each His in D


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Inorganic Chemistry


Table 3. Stability Constants (log β Values), pKa Values and Conditional Binding Constants (CI7.4) for Cu(II) Complexes of Aβ4−9, Aβ11−16, pAβ11−16, and Aβ12−16 Peptides at I = 0.1 M (KNO3) Determined by Potentiometry at 25 °Ca

detected by potentiometry. EPR spectroscopy yielded two additional species in the pH range 15).46 Concomitantly, the 4N complex is formed immediately from the 2N complexes. Thus, the anchoring of the Cu(II) ion at His13 is more likely, or even dominant. Further support for this concept can be obtained from the analysis of conditional dissociation constants (CI7.4)75,76 presented in the Discussion. Acid−Base and Complex-Forming Properties of VHHQK-am (Aβ12−16). The VHHQK-am peptide has four exchangeable hydrogen ions, at two His imidazoles, and Val12

therefore be similar to those containing a single His residue further up the peptide chain.48 This prediction was confirmed by spectroscopic experiments that demonstrated the presence of multiple pH-dependent Cu(II) complex species (Figure 4, Figures S3−S4). In this instance, all species detected by EPR spectroscopy could be assigned to complexes also identified by CD and UV−vis. The absence of isosbestic and isodichroic points in these titrations confirms an overlap of several complexes in the whole pH range tested. Altogether, six protonic forms were found for Cu(pAβ11−16) complexes (Table 2). The most protonated one, CuH2L, appears at low pH and is a 1N complex in which the Cu(II) ion is anchored to the imidazole of His13 or His14. This complex can be seen as a shoulder in absorption bands around pH 5. Its weak absorption around 750 nm precludes formation of an isosbestic point between the aqua ion and a 2N species. The next deprotonation yields CuHL, a complex exhibiting a 2N coordination by nitrogen atoms of both imidazole side chains. It is characterized by the weak absorption band at 650 nm (Figure 4A, Table 2) and EPR parameters that support the assignment of a 2N2O coordination sphere (Figure 4C, Table 2). The CuH2L and CuHL complexes are principally inactive in CD, due to a large distance, and thus a small overlap, of the Cu(II) chromophore with the chiral Cα moiety. The only band in CD spectra related to these complexes is seen around 250− 255 nm and can be attributed to the NIm to Cu(II) charge transfer band (Figure 4B).46,72−74 The CuHL complex, which is relatively highly abundant at pH 5−6, is replaced by a minor consecutive species, CuL. The CD spectrum of this latter complex is similar to that of CuHL, while its absorption band is blue-shifted toward ∼600 nm. The clear correlation between the potentiometric species and the spectral features (Figure 4D, Figure S4) suggests that no amide nitrogen coordination is present in this species. Then the only possibility is a deprotonation of coordinated water molecule. The next F


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Inorganic Chemistry


of fluorescence of Aβ4−12 by ca. 60% (Figure 6), which is typical for CuAβ complexes.50,78 The Aβ12−16 peptide and its

amine and Lys16 amino groups. The pKa difference between His13 and His14 is 0.68; thus these residues are practically equivalent. The average pKa, 6.07, is the most acidic of all four peptides studied, as a result of the highest concentration of the positive charge (four positive charges per five residues). Also the next two macroconstants, essentially equivalent to amino group constants of Val12 and Lys16, respectively, are slightly more acidic than in other peptides. VHHQK-am has a unique arrangement of N-terminal residues, as Xxx-His-Yyy and Xxx-Yyy-His sequences can form competitive 3N and 4N chelate ring systems, respectively.77 The CuHL complex, the first observed complex species, is a 3N complex with the characteristic absorption band at 591 nm. This complex is formed directly from the Cu(II) aqua ion as evidenced by an isosbestic point at 760 nm, with a pKa of 3.56 (Figure 5A; Figure S5). EPR spectroscopy yielded an additional species beginning at pH < 3 and persisting up to pH ≈ 4.2 (Figure 5C), most likely with 1N3O coordination. This species was not detected by other room temperature methods and therefore attributed to a freezinginduced artifact. The CuHL complex is replaced by the CuL complex (pKa ≈ 5.0; Figure S6), which has the ATCUN/NTS 4N coordination, as confirmed by the fingerprint CD (Figure 5B) and EPR (Figure 5C) spectral patterns (Table 2). Stoichiometric considerations allow us to state that this species contains protonated His13 and Lys16 residues, and their respective deprotonations yield the CuH−1L and CuH−2L complexes. The excellent match of spectroscopic and potentiometric titrations (Figure 5D) confirms this analysis. Kinetics of Cu(II) Exchange between Aβ4−x and Aβ12−x. To examine the rate of Cu(II) transfer between the two highest affinity N-truncated peptides, Aβ4−12 was used in place of Aβ4−9 in order to exploit the Tyr fluorescence as a reporter probe. Copper coordination resulted in the reduction

Figure 6. Time-dependence of Tyr10 fluorescence during Cu(II) transfer between CuAβ4−12 and Aβ12−16. The system was equilibrating thermally for about 60 min. Pseudo-first-order fits of the Cu(II) exchange reactions between Aβ4−12 and Aβ12−16 are shown as dashed black lines.

Cu(II) complex had no fluorescence. In one instance, Aβ4−12 was added to the preformed CuAβ12−16 complex, while, in another, the Aβ12−16 peptide was added to the preformed CuAβ4−12 complex. The fluorescence was monitored for at least 48 h incubation at 25 °C. The fluorescence increased in time for CuAβ4−12 after adding 1 equiv of Aβ12−16, corresponding to the Cu(II) transfer from the CuAβ4−12 complex to Aβ12−16. In the reversed order experiment when Aβ4−12 was added to CuAβ12−16, the signal decreased in comparison to the control, indicating the Cu(II)induced quenching of Tyr10 in Aβ4−12 resulting from the Cu(II) transfer. Thus, both peptides can withdraw the Cu(II) G


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Inorganic Chemistry ions from each other, depending on which free form is in excess, until equilibrium is reached. This behavior is consistent with the comparable femtomolar Cu(II) dissociation constants of Aβ12−16 and Aβ4−12 peptides. The reaction was very slow, with the half-lives of Cu(II) release from CuAβ4−12 and CuAβ12−16 being 24 and 16 h, respectively. The pseudo-first-order fitting of the reaction course could also be used to verify the ratio of Cu(II) affinities of these two peptides, by establishing the equilibrium fluorescence. Considering the fact that the fluorescence is the sum of contributions of the free Aβ4−12 peptide (0.76 RFU) and its Cu(II) complex (0.32 RFU), and that the fluorescence intensities plateau at 0.50 RFU and 0.48 RFU for the Cu(II) release from CuAβ4−12 and CuAβ12−16, respectively, we obtain the average equilibrium state of 61 ± 2% of Cu(II) bound to Aβ4−12. Taking into account the concentrations of interacting species this yields the ratio of dissociation constants of 0.46; with Kd = 9.4 fM for Aβ12−16, the Kd = 4.3 fM is obtained for CuAβ4−12. Assuming a purely dissociative mechanism of Cu(II) transfer, a ca. 2-fold higher Cu(II) affinity of Aβ4−12 is consistent with a slower Cu(II) release. Although the dissociation constant for Aβ4−12 is determined with a low accuracy, one may consider Aβ4−12 to be sufficiently similar to Aβ4−9.

Figure 7. (A) 4N coordination mode of Aβ4−9, (B) 4N coordination mode of Aβ11−16, (C) 4N coordination mode of pAβ11−16, (D) 3N coordination mode of Aβ12−16, (E) 4N coordination mode of Aβ12−16. A full description of all pH-dependent coordination spheres (CuHmLn) is given in Table S2.

constants, were fully corroborated by three independent spectroscopic techniques. Although it has His residues in position three and four, the pAβ11−16 peptide was demonstrated to coordinate the Cu(II) in a different fashion, with 1N, 2N, and 4N species being formed (Table 2). The loss of cooperativity in the formation of fused chelate rings was due to the absence of the free amine function at its N-terminus, which serves as the second anchor for the metal ion. The peptide Aβ12−16 has His residues in the second and third position, enabling it to anchor Cu(II) via two coordination modes. In addition to the 4N ATCUN/NTS motif (Figure 7E), it can use His in the second position as an alternative anchor to create a 3N coordination mode (Figure 7D). Both binding modes have been realized in a pH dependent fashion. The 3N complex was formed at acidic pH and was converted into the typical 4N complex with the pKa ≈ 5. The latter complex proved more stable at alkaline pH where the free energy gain from coordination of an extra peptide nitrogen exceeded the penalty of its deprotonation. Similar phenomena were presented recently for model peptides AHH and AHH−am.77 Cu(II) Affinities and Exchange between the Low Molecular Weight Products of Aβ Hydrolysis. The values of CI7.4 presented in Table 3 are equivalent to conditional Cu(II) dissociation constants of the complexes (or variously protonated sets of complexes) of given peptides at this pH.75,76 As shown in the bottom line of Table 3, Aβ4−9 formed the strongest complex, followed closely by Aβ12−16. The third ATCUN/NTS motif peptide, Aβ11−16 is ca. 200-fold weaker. While the reasons for differences in stabilities of ATCUN/ NTS complexes have not been fully explained, there is a partial correlation between the basicities of coordinated peptide nitrogens and the complex stabilities.69,82,83 The pAβ11−16 peptide forms by far the weakest complex, due to the absence of the anchoring N-terminal amine function, more than 3000 times weaker than its parent peptide. The apparent abundance of Aβ4−42 and presumably its C-terminally truncated analogues, which have fM Cu(II) affinities, as demonstrated here and before,50 makes the pyroglutamate form of Aβ11−x an unlikely Cu(II) ligand in vivo. Nevertheless, it is worth noting that the

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DISCUSSION Coordination Properties of the Studied Peptides. Three out of four peptides studied in this work carry ATCUN/ NTS sites which enable high affinity Cu(II) binding. The peptide Aβ4−9 contains a single His in position 3, and the peptide Aβ11−16 has His residues in positions three and four. The ATCUN/NTS paradigm requires that the whole coordination process occurs within the first three N-terminal residues, and the identity of the residues upstream of the Cu(II)-anchoring His in position 3 is largely irrelevant for the coordination mode (although it may influence the affinity).46−50 The data presented above confirm this paradigm. Each peptide exhibits only a single spectroscopic complex signature at pH > 7, very similar to each other. The alternate pattern of d−d component bands in CD, together with other spectroscopic parameters, clearly indicates the formation of 4N complexes at physiological pH (Figure 7). The Cu(II) binding to longer Aβ11−x peptides, Aβ11−20 and Aβ11−28, was studied before using the same methodology, with significantly higher Cu(II) stability constants and significantly lower peptide function basicities being reported.79,80 However, while spectroscopic parameters of respective complexes were determined, the pH distribution of these parameters was not compared with the potentiometric results. Our results have been thoroughly validated, and therefore we do not include the other data in further discussions. A conditional dissociation constant at pH 7.4 was published for a similar peptide, Aβ11−15.81 The published value, Kd = 34 ± 5 fM, is >50-fold lower than that obtained by us for the Aβ11−16 peptide. This difference cannot be attributed to the presence of Lys16, as evidenced by vast literature on ATCUN/NTS complexes and the authors’ comparable result (10 ± 3 fM) for the aggregation-prone Aβ11−40.81 The discrepancy probably stems from a different methodology, which relied on competitive titrations of glycine and histidine.81 We applied rigorously controlled potentiometric titrations and the species distributions, which are in direct relationship with the stability H


Inorganic Chemistry

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ACKNOWLEDGMENTS The study was sponsored by the National Science Centre of Poland, Grant No. 2014/15/B/ST5/05229 (W.B.). The equipment used was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by the European Regional Development Fund and Innovative Economy, The National Cohesion Strategy of Poland. S.C.D. was supported by a fellowship awarded by the faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne.

pGlu residue appears to be a better Cu(II) binding site than a typical peptide nitrogen; e.g., the binding affinity of the second Cu(II) equivalent to Aβ4−16 is >30-fold lower than the binding affinity of one Cu(II) equivalent to pAβ11−16.50 The Cu(II) exchange reaction between the ATCUN/NTS motifs studied here was found to be very slow, occurring over the course of several days (Figure 6). The time scales of various aspects of brain extracellular biochemistry range from the millisecond periodicity of neurotransmitter pulses across the synaptic cleft, seconds/minutes timescale of neuromodulator action, to the 6 h period of renewal of cerebrospinal fluid.84 The time of equilibration of Cu(II) ions between the FRH and VHH sites is ca. 10-fold slower from the slowest of these processes. Therefore, we may conclude that high affinity Cu(II) complexes with ATCUN/NTS peptide products of NEP, Aβ4−9, Aβ12−x , or other Aβ 4−x , (Kd of 30 fM demonstrated for Aβ4−16)50 are so inert in terms of Cu(II) release in nonredox exchange processes that they should be considered as coexistent and unreactive within time windows of their putative bioactivity. As demonstrated for Aβ4−16, such complexes are also highly resistant to reduction-driven exchange with metallothionein-3 or low molecular weight reductants.55,85 The slow rate of Cu(II) equilibration between Aβ4−9 and Aβ12−16 is consistent with an essentially dissociative mechanism of the exchange, analogous to the Cu(II) transfer from human serum albumin to the N-terminus of hepcidin.86

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CONCLUSION Our results indicate that multiple short products of Aβ peptide digestion can carry/control Cu(II) ions present in the extracellular compartments in the brain. Once these complexes are formed, they are unlikely to exchange Cu(II) with other extracellular ligands on a physiological time scale. It is currently unknown whether Cu(II) binding by Aβ4−9 and other Ntruncated Aβ peptides occurs in vivo,17 although this remains an important question in order to clarify whether such lowmolecular-weight, soluble complexes could participate in brain copper physiology and if a shift toward more insoluble Aβ4−42 species, especially in the hippocampus, may lead to a loss of function or gain of toxic function.56 ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b03051.

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Additional analyses of UV−vis, CD, and potentiometric titrations (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (W.B.). *E-mail: [email protected] (S.C.D.). ORCID

Simon C. Drew: 0000-0002-1459-9865 Wojciech Bal: 0000-0003-3780-083X Author Contributions #

K.B.A. and M.M. contributed equally.

Notes

The authors declare no competing financial interest. I


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