TY - JOUR
AU - Makarov, A A
AB - Abstract Interaction of Zn2+ with the metal-binding domain of the English (H6R) amyloid-β mutant results in the formation of peptide dimers. The mutation causes the exclusion of His6 from the zinc chelation pattern observed in the intact domain and triggers the assembly of the dimers via zinc ions coordinated by 11EVHH14 fragments. Graphical Abstract Open in new tabDownload slide The English familial mutation causes the exclusion of His6 from the zinc chelation pattern and triggers the assembly of Aβ1–16 dimers. Alzheimer's disease (AD), the most common type of dementia, is associated with oligomerization and aggregation of amyloid-β peptide (Aβ).1 There are several familial mutations located within Aβ that cause early onset AD: English (H6R), Tottori (D7N), Taiwan (D7H), Flemish (A21G), Dutch (E22Q), Italian (E22K), Arctic (E22G), and Iowa (D23N).2,3 Pathological Aβ aggregation is mediated by zinc ions,4–7 therefore the English familial AD mutation (H6R),3 which affects the His6 residue from zinc coordination sphere of the metal-binding domain (residues 1–16, Aβ1–16) of Aβ,8 is of particular interest. It has been shown that English mutation increases neurotoxicity of respective Aβ (H6R-Aβ) oligomers9 and accelerates H6R-Aβ fibril formation.10 However, the impact of zinc ions on the aggregation state of H6R-Aβ has not yet been investigated. Synthetic peptides corresponding to Aβ1–16 are good soluble models to examine zinc binding to Aβ.8,11–15 In the present work, we have characterized the interaction of zinc ions with the H6R mutant of Aβ1–16 amidated at the C-terminus, with either acetylated (AcH6R-Aβ1–16) or free (H6R-Aβ1–16) N-terminus, using surface plasmon resonance (SPR) biosensing, isothermal titration calorimetry (ITC) and NMR spectroscopy. The ability of AcH6R-Aβ1–16 to form oligomers in the presence of zinc ions has been tested using SPR biosensing. This has been done by registering interactions between the dissolved AcH6R-Aβ1–16 peptides and the same peptides immobilized on an optical chip. A set of sensograms has been obtained corresponding to the injections of solution with different concentrations of AcH6R-Aβ1–16 in the presence and absence of zinc ions (Fig. 1, details provided in the ESI†). The results given in Fig. 1 indicate that oligomers are formed only in the presence of zinc ions. Fig. 1 Open in new tabDownload slide SPR sensograms of AcH6R-Aβ1–16 (analyte) interactions with the immobilized ligand (AcH6R-Aβ1–16) at 25 °C in 10 mM HEPES, in the presence and absence of 0.1 mM ZnCl2, pH 6.8. Total analyte concentrations in the running buffer were 2, 5, 10, 15 and 20 μM. The stoichiometry of Zn2+ binding to AcH6R-Aβ1–16 determined by ITC (Fig. 2B, N = 0.5) suggests the formation of a complex where two peptide molecules interact with one zinc ion. In contrast, non-modified AcAβ1–16 forms a monomeric complex in the presence of zinc ions with a stoichiometry of 1 : 1 of the peptide–metal interaction (Fig. 2A).11,12 This process can be described by the following equilibrium scheme,16,17 where P denotes the peptide AcH6R-Aβ1–16: Zn+P→K1Zn⋅P1 P+Zn⋅P→K2P⋅Zn⋅P2 Fig. 2 Open in new tabDownload slide ITC titration curves (upper panel) and the binding isotherms (lower panel) for the zinc interactions with AcAβ1–1612 (A) and AcH6R-Aβ1–16 (B) at 25 °C in 50 mM Tris buffer, pH 7.3. By fitting the binding isotherm, the value of apparent association constant Ka = K1 × K2 = 0.24 × 104 M−2 was determined. In order to rule out the participation of the N-terminal amino group in chelation of zinc ions, we have compared the thermodynamic parameters of Zn2+-binding to the peptides with acetylated (AcH6R-Aβ1–16) and free N-terminus (H6R-Aβ1–16) (Table S1, ESI†). Both peptides bind Zn2+ similarly, indicating that the N-terminus of H6R-Aβ1–16 does not participate in Zn2+ chelation. Formation of the dimer Zn·(AcH6R-Aβ1–16)2 in solution was unambiguously confirmed by NMR. Sharp signals observed in NMR spectra of the peptide AcH6R-Aβ1–16 (Fig. 3A, Table S1, ESI†) are broadened upon addition of zinc ions (Fig. 3B, Table S2, ESI†), which is typical for the formation of zinc complexes with Aβ fragments containing the metal binding domain.18–20 However, in contrast to other previous studies of Aβ peptides,11,13,19 interaction of AcH6R-Aβ1–16 with Zn2+ leads to the appearance of the additional set of the peptide resonances (see assignments in Table S3, ESI†). For example, in addition to signals at 0.76 and 0.87 ppm which correspond to the methyl groups of Val12 of Zn·AcH6R-Aβ1–16 (Table S2, ESI†), a new signal appears at 0.23 ppm (Fig. 3B, Table S3, ESI†). This signal along with the resonances from several other residues (Table S3, ESI†) belongs to the dimeric form of the complex Zn·(AcH6R-Aβ1–16)2. Indeed, an increase of the total peptide concentration results in higher intensities of the signals assigned to the dimeric form (Fig. S2, ESI†). Moreover, Job's plot for intensity changes of the signal at 0.23 ppm (Fig. 3B) observed in experiments carried out using the method of continuous variations21 (details provided in the ESI†) indicates that the stoichiometry of peptide–zinc interaction is 2 : 1 (Fig. 4, curve 1). Meanwhile Job's plot of the changes in His13 and His14 Hε1 chemical shifts of the monomeric Zn·(AcH6R-Aβ1–16) form (Fig. 4, curve 2) shows a 1 : 1 stoichiometry of the peptide–zinc interaction. Fig. 3 Open in new tabDownload slide Representative parts of the 1H NMR spectra of the 1.1 mM AcH6R-Aβ1–16 peptide (denoted P) in the free state (A) and in the presence of a two-fold molar excess of ZnCl2 (B). Root-mean-square deviations (RSMD) of the 1H and 13C chemical shifts between the free and zinc-bound states of AcH6R-Aβ1–16 (C). Data for the peptide monomer Zn·P are shown in black, and for the dimer P·Zn·P in grey. Experiments were carried out in 10 mM Bis–Tris-d19 buffer in D2O, pH 6.8, 283 K and 600 MHz. Fig. 4 Open in new tabDownload slide Results of the Zn2+ isomolar NMR titration of AcH6R-Aβ1–16. Abscissa represents the mole fraction of ZnCl2, and the ordinate axis shows the product of ΔX·[P]0, where ΔX is either ΔI (change of the relative intensity of the Val12 Hγ* signal at 0.2 ppm) for the curve 1 (open circles), or Δδ (change of the chemical shift of histidine Hδ2 signals) for the curve 2 (black circles), and [P]0 – represents the total AcH6R-Aβ1–16 peptide concentration in the sample. Curve 2 corresponds to the formation of a zinc–peptide complex with the stoichiometry 1 : 1. Curve 1 corresponds to the formation of the peptide dimer in complex with a single zinc atom (stoichiometry 2 : 1). Experimental details are given in the ESI.† Chemical shift changes measured in Zn2+ titration NMR experiments (Fig. S3–S6, ESI†) were used to identify metal binding sites both in monomeric Zn·(AcH6R-Aβ1–16) and dimeric Zn·(AcH6R-Aβ1–16)2 complexes (Fig. 3C). Observed changes highlight the group of residues involved in the binding of zinc ions in the monomeric complex (Fig. 3C, black bars). Such a binding site is formed by two histidine residues (His13 and His14) and Glu11. It should be noted that these three residues compose the primary zinc-recognition fragment 11EVHH14.12 Changes in the chemical shifts that accompany the formation of the dimeric complex are much higher than those observed for monomers (Fig. 3C, grey bars). They indicate that the zinc interaction interface in the Zn·(AcH6R-Aβ1–16)2 complex is also formed by the fragment, 11EVHH14, where the most probable zinc ion chelators are Glu11 and His14 of the two interacting subunits. At the same time considerable chemical shift changes of the residues that are unable to bind zinc ions (i.e. Tyr10 and Val12) are observed. Such changes can only be explained by the conformational reorganization of the peptide molecule upon formation of a stable dimeric complex. Exchange cross-peaks in the 2D ROESY spectrum (Fig. S7, ESI†) between the resonances that belong to monomeric and dimeric peptide complexes indicate that there is an equilibrium between the two forms. For example, such exchange peaks with positive intensities are seen between the Hγ* signals of Val12 at 0.21 and 0.84 ppm. However, the existence of two separate sets of signals corresponding to the Zn·(AcH6R-Aβ1–16) and Zn·(AcH6R-Aβ1–16)2 complexes evidences that the dimeric complex is stable on the NMR time scale in contrast to all other studied zinc-bound Aβ1–16 dimers.11,13,19 Comparison of the NMR spectra of AcH6R-Aβ1–16 and H6R-Aβ1–16 recorded under identical conditions in the presence of ZnCl2 (Fig. S8 and S9, ESI†) confirms our ITC data (Table S1, ESI†) showing that the N-terminal amino group does not participate in interaction with zinc ions at neutral pH. Previously it was shown that complexes of Aβ1–16 with zinc ions exist as either dimers,22 where residues from the 11EVHH14 regions of two interacting peptide chains jointly coordinate the zinc ion, or monomers.11,12 In monomers zinc ions are coordinated by three residues from the region 11EVHH14 together with the distant His6 residue of the Aβ1–16 molecule. The first possibility can be realized in the pathogenic dimers of full-length Aβ molecules,23,24 while the existence of soluble monomeric Aβ complexes with one zinc ion can be associated with a normal physiological state of Aβ in biological fluids. Recently we have shown that withdrawal of His6 from zinc ion chelation in conjunction with the 11EVHH14 site upon Ser8 phosphorylation leads to zinc-dependent dimerization of the respective Aβ1–16 peptide (pS8-Aβ1–16).13 In the present study we have for the first time documented the formation of water-soluble zinc-bonded dimers of AcH6R-Aβ1–16. NMR spectra of human AcAβ1–16 in the presence of Zn2+ ions highlight the formation of dimers structurally similar to Zn·(AcH6R-Aβ1–16)2, as evidenced by characteristic resonances of the Val12 methyl group and His14 HN (Fig. S10, ESI†). However, the quantity of a dimer formed by the wild-type peptide is substantially smaller compared to the H6R analog, a result which can be explained by the involvement of the His6 residue in coordination of zinc ions in the monomeric peptide–zinc complex formed by AcAβ1–16. The dimerization interface in AcH6R-Aβ1–16 is virtually identical to that observed for Zn·(pS8-Aβ1–16)2.13 Each of the peptides AcH6R-Aβ1–16 and pS8-Aβ1–16 has only one amino acid change (due to mutation or chemical modification) distinguishing their sequences from the native Aβ1–16 form. Although the changes have different origins (genetic versus kinase dependent) they result in the formation of zinc-bound dimers stabilized by the same dimerization interface as in the intact Aβ1–16,22 suggesting a general mechanism of zinc-bound Aβ dimer formation. Conclusions We have demonstrated that the interaction of the metal-binding domain of the English (H6R) Aβ mutant with zinc ions results in the formation of stable soluble dimers under physiologically relevant conditions. These findings suggest that the possible molecular mechanism of the AD pathogenicity of English mutation could be related to the increased propensity of respective Aβ species to undergo zinc-dependent oligomerization. The interface of zinc-mediated complexes is formed by the residues 11EVHH14 from the two AcH6R-Aβ1–16 chains and appears to be identical to other zinc-induced Aβ dimers,13,16,22,23 thus making the Aβ site 11–14 a putative drug target to prevent zinc-induced aggregation of Aβ in AD progression. Acknowledgements This work was supported by the Russian Foundation for Basic Research (grant 13-04-40108-comfi), and by the Ministry of Education and Science of the Russian Federation (agreement no. 14.Z50.31.0014, Contract No. 14.604.21.0074, ID RFMEFI60414X0074). 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Kozin, A. A. Kulikova and A. N. Istrate are authors that made equal contributions to this manuscript. © The Royal Society of Chemistry 2015 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © The Royal Society of Chemistry 2015
TI - The English (H6R) familial Alzheimer's disease mutation facilitates zinc-induced dimerization of the amyloid-β metal-binding domain
JF - Metallomics
DO - 10.1039/c4mt00259h
DA - 2015-03-11
UR - https://www.deepdyve.com/lp/oxford-university-press/the-english-h6r-familial-alzheimer-s-disease-mutation-facilitates-zinc-0KxhLlWniM
SP - 422
EP - 425
VL - 7
IS - 3
DP - DeepDyve
ER -