Modeling Copper Binding to the Amyloid‑β Peptide at Different pH: Toward a Molecular Mechanism for Cu Reduction

Oxidative stress, including the production of reactive oxygen species (ROS), has been reported to be a key event in the etiology of Alzheimer’s disease (AD). Cu has been found in high concentrations in amyloid plaques, a hallmark of AD, where it is bound to the main constituent amyloid-β (Aβ) peptide. Whereas it has been proposed that Cu-Aβ complexes catalyze the production of ROS via redox-cycling between the Cu­(I) and Cu­(II) state, the redox chemistry of Cu-Aβ and the precise mechanism of redox reactions are still unclear. Because experiments indicate different coordination environments for Cu­(II) and Cu­(I), it is expected that the electron is not transferred between Cu-Aβ and reactants in a straightforward manner but involves structural rearrangement. In this work the structures indicated by experimental data are modeled at the level of modern density-functional theory approximations. Possible pathways for Cu­(II) reduction in different coordination sites are investigated by means of first-principles molecular dynamics simulations in the water solvent and at room temperature. The models of the ligand reorganization around Cu allow the proposal of a preferential mechanism for Cu-Aβ complex reduction at physiological pH. Models reveal that for efficient reduction the deprotonated amide N in the Ala 2-Glu 3 peptide bond has to be protonated and that interactions in the second coordination sphere make important contributions to the reductive pathway, in particular the interaction between COO^– and NH₂ groups of Asp 1. The proposed mechanism is an important step forward to a clear understanding of the redox chemistry of Cu-Aβ, a difficult task for spectroscopic approaches as the Cu-peptide interactions are weak and dynamical in nature

Dynamics of Zn^II Binding as a Key Feature in the Formation of Amyloid Fibrils by Aβ11-28

Supramolecular assembly of peptides and proteins into amyloid fibrils is of multifold interest, going from materials science to physiopathology. The binding of metal ions to amyloidogenic peptides is associated with several amyloid diseases, and amyloids with incorporated metal ions are of interest in nanotechnology. Understanding the mechanisms of amyloid formation and the role of metal ions can improve strategies toward the prevention of this process and enable potential applications in nanotechnology. Here, studies on Zn^II binding to the amyloidogenic peptide Aβ11-28 are reported. Zn^II modulates the Aβ11-28 aggregation, in terms of kinetics and fibril structures. Structural studies suggest that Aβ11-28 binds Zn^II by amino acid residues Glu11 and His14 and that Zn^II is rapidly exchanged between peptides. Structural and aggregation data indicate that Zn^II binding induces the formation of the dimeric Zn^II₁(Aβ11-28)₂ species, which is the building block of fibrillar aggregates and explains why Zn^II binding accelerates Aβ11-28 aggregation. Moreover, transient Zn^II binding, even briefly, was enough to promote fibril formation, but the final structure resembled that of apo-Aβ11-28 amyloids. Also, seeding experiments, i.e., the addition of fibrillar Zn^II₁(Aβ11-28)₂ to the apo-Aβ11-28 peptide, induced aggregation but not propagation of the Zn^II₁(Aβ11-28)₂-type fibrils. This can be explained by the dynamic Zn^II binding between soluble and aggregated Aβ11-28. As a consequence, dynamic Zn^II binding has a strong impact on the aggregation behavior of the Aβ11-28 peptide and might be a relevant and so far little regarded parameter in other systems of metal ions and amyloidogenic peptides

Identifying, By First-Principles Simulations, Cu[Amyloid-β] Species Making Fenton-Type Reactions in Alzheimer’s Disease

According to the amyloid cascade hypothesis, amyloid-β peptides (Aβ) play a causative role in Alzheimer’s disease (AD), of which oligomeric forms are proposed to be the most neurotoxic by provoking oxidative stress. Copper ions seem to play an important role as they are bound to Aβ in amyloid plaques, a hallmark of AD. Moreover, Cu–Aβ complexes are able to catalyze the production of hydrogen peroxide and hydroxyl radicals, and oligomeric Cu–Aβ was reported to be more reactive. The flexibility of the unstructured Aβ peptide leads to the formation of a multitude of different forms of both Cu­(I) and Cu­(II) complexes. This raised the question of the structure–function relationship. We address this question for the biologically relevant Fenton-type reaction. Computational models for the Cu–Aβ complex in monomeric and dimeric forms were built, and their redox behavior was analyzed together with their reactivity with peroxide. A set of 16 configurations of Cu–Aβ was studied and the configurations were classified into 3 groups: (A) configurations that evolve into a linearly bound and nonreactive Cu­(I) coordination; (B) reactive configurations without large reorganization between the two Cu redox states; and (C) reactive configurations with an open structure in the Cu­(I)–Aβ coordination, which have high water accessibility to Cu. All the structures that showed high reactivity with H₂O₂ (to form HO^•) fall into class C. This means that within all the possible configurations, only some pools are able to produce efficiently the deleterious HO^•, while the other pools are more inert. The characteristics of highly reactive configurations consist of a N–Cu­(I)–N coordination with an angle far from 180° and high water crowding at the open side. This allows the side-on entrance of H₂O₂ and its cleavage to form a hydroxyl radical. Interestingly, the reactive Cu­(I)–Aβ states originated mostly from the dimeric starting models, in agreement with the higher reactivity of oligomers. Our study gives a rationale for the Fenton-type reactivity of Cu–Aβ and how dimeric Cu–Aβ could lead to a higher reactivity. This opens a new therapeutic angle of attack against Cu–Aβ-based reactive oxygen species production

Measurement of Interpeptidic Cu(II) Exchange Rate Constants by Static Fluorescence Quenching of Tryptophan

The interpeptidic exchange of Cu­(II) between biologically relevant peptides like Gly-His-Lys (GHK) was measured through proximity static fluorescence quenching of a noncoordinating tryptophan residue by Cu­(II). The inability to spectrally distinguish between starting and final Cu­(H_–1GHK)⁺ complexes by the current methods was solved by the replacement of noncoordinating lysine for tryptophan in the starting complex, Cu­(H_–1GHW). Because the apoGHW is the only fluorescent species, the recovered fluorescence is directly proportional to the [Cu­(II)]_exchanged between GHW and GHK. The apparent second-order rate constants of the exchanges from Cu­(H_–1GHW) to GHK and DAHK are 1.6 (±0.2) × 10² and 5.0 (±0.7) × 10¹ M^–1 s^–1, respectively. The easy-to-implement kinetic fluorescent method described here for Cu­(II) interpeptidic exchange can be expanded to other biological systems

Insights into the Mechanisms of Amyloid Formation of Zn^II-Ab11-28: pH-Dependent Zinc Coordination and Overall Charge as Key Parameters for Kinetics and the Structure of Zn^II-Ab11-28 Aggregates

Self-assembly of amyloidogenic peptides and their metal complexes are of multiple interest including their association with several neurological diseases. Therefore, a better understanding of the role of metal ions in the aggregation process is of broad interest. We report pH-dependent structural and aggregation studies on Zn^II binding to the amyloidogenic peptide Ab11-28. The results suggest that coordination of the N-terminal amine to Zn^II is responsible for the inhibition of amyloid formation and the overall charge for amorphous aggregates

Copper Coordination to Native N‑Terminally Modified versus Full-Length Amyloid-β: Second-Sphere Effects Determine the Species Present at Physiological pH

Alzheimer’s disease is characterized by senile plaques in which metallic ions (copper, zinc, and iron) are colocalized with amyloid-β peptides of different sequences in aggregated forms. In addition to the full-length peptides (Aβ1-40/42), N-terminally truncated Aβ3-40/42 forms and their pyroglutamate counterparts, Aβp3-40/42, have been proposed to play key features in the aggregation process, leading to the senile plaques. Furthermore, they have been shown to be more toxic than the full-length Aβ, which made them central targets for therapeutic approaches. In order to better disentangle the possible role of metallic ions in the aggregation process, copper­(II) coordination to the full-length amyloid peptides has been extensively studied in the last years. However, regarding the N-terminally modified forms at position 3, very little is known. Therefore, copper­(I) and copper­(II) coordination to those peptides have been investigated in the present report using a variety of complementary techniques and as a function of pH. Copper­(I) coordination is not affected by the N-terminal modifications. In contrast, copper­(II) coordination is different from that previously reported for the full-length peptide. In the case of the pyroglutamate form, this is due to preclusion of N-terminal amine binding. In the case of the N-terminally truncated form, alteration in copper­(II) coordination is caused by second-sphere effects that impact the first binding shell and the pH-dependent repartition of the various [Cu­(peptide)] complexes. Such second-sphere effects are anticipated to apply to a variety of metal ions and peptides, and their importance on changing the first binding shell has not been fully recognized yet

Cu(II) Affinity for the Alzheimer’s Peptide: Tyrosine Fluorescence Studies Revisited

Copper­(II) binding to the amyloid-β peptide has been proposed to be a key event in the cascade leading to Alzheimer’s disease. As a direct consequence, the strength of the Cu­(II) to Aβ interaction, that is, the Cu­(II) affinity of Aβ, is a very important parameter to determine. Because Aβ peptide contain one Tyr fluorophore in its sequence and because Cu­(II) does quench Tyr fluorescence, fluorescence measurements appear to be a straightforward way to obtain this parameter. However, this proved to be wrong, mainly because of data misinterpretation in some previous studies that leads to a conflicting situation. In the present paper, we have investigated in details a large set of fluorescence data that were analyzed with a new method taking into account the presence of two Cu­(II) sites and the inner-filter effect. This leads to reinterpretation of the published data and to the determination of a unified affinity value in the 10¹⁰ M^–1 range

pH-Dependent Cu(II) Coordination to Amyloid-β Peptide: Impact of Sequence Alterations, Including the H6R and D7N Familial Mutations.

Copper ions have been proposed to intervene in deleterious processes linked to the development of Alzheimer’s disease (AD). As a direct consequence, delineating how Cu(II) can be bound to amyloid-β (Aβ) peptide, the amyloidogenic peptide encountered in AD, is of paramount importance. Two different forms of [Cu^II(Aβ)] complexes are present near physiological pH, usually noted components <b>I</b> and <b>II</b>, the nature of which is still widely debated in the literature, especially for <b>II</b>. In the present report, the phenomenological pH-dependent study of Cu(II) coordination to Aβ and to ten mutants by EPR, CD, and NMR techniques is described. Although only indirect insights can be obtained from the study of Cu(II) binding to mutated peptides, they reveal very useful for better defining Cu(II) coordination sites in the native Aβ peptide. Four components were identified between pH 6 and 12, namely, components <b>I</b>, <b>II</b>, <b>III</b> and <b>IV</b>, in which the predominant Cu(II) equatorial sites are {−NH₂, CO (Asp1–Ala2), N_im (His6), N_im (His13 or His14)}, {−NH₂, N^– (Asp1–Ala2), CO (Ala2–Glu3), N_im}, {−NH₂, N^– (Asp1–Ala2), N^– (Ala2–Glu3), N_im} and {−NH₂, N^– (Asp1–Ala2), N^– (Ala2–Glu3), N^– (Glu3–Phe4)}, respectively, in line with classical pH-induced deprotonation of the peptide backbone encountered in Cu(II) peptidic complexes formation. The structure proposed for component <b>II</b> is discussed with respect to another coordination model reported in the literature, that is, {CO (Ala2–Glu3), 3 N_im}. Cu(II) binding to the H6R-Aβ and D7N-Aβ peptides, where the familial H6R and D7N mutations have been linked to early onset of AD, has also been investigated. In case of the H6R mutation, some different structural features (compared to those encountered in the native [Cu^II(Aβ)] species) have been evidenced and are anticipated to be important for the aggregating properties of the H6R-Aβ peptide in presence of Cu(II)

Concept for Simultaneous and Specific in Situ Monitoring of Amyloid Oligomers and Fibrils via Förster Resonance Energy Transfer

Oligomeric species of amyloidogenic peptides or proteins are often considered as the most toxic species in several amyloid disorders, like Alzheimer or Parkinson’s diseases, and hence came into the focus of research interest and as a therapeutic target. An easy and specific monitoring of oligomeric species would be of high utility in the field, as it is the case for thioflavin T fluorescence for the fibrillar aggregates. Here, we show proof of concept for a new sensitive method to increase specific detection of oligomers by two extrinsic fluorophores. This is achieved by exploiting a Förster resonance energy transfer (FRET) between the two fluorophores. Thus, a mixture of two extrinsic fluorophores, bis-ANS and a styrylquinoxalin derivative, enabled one to monitor simultaneously and in situ the presence of oligomers and fibrils of amyloidogenic peptides. Thereby, the formation of oligomers and their transformation into fibrils can be followed

Impact of Cu(II) Binding on Structures and Dynamics of Aβ₄₂ Monomer and Dimer: Molecular Dynamics Study

The classical force field, which is compatible with the Amber force field 99SB, has been obtained for the interaction of Cu­(II) with monomer and dimers of amyloid-β peptides using the coordination where Cu­(II) is bound to His6, His13 (or His14), and Asp1 with distorted planar geometry. The newly developed force field and molecular dynamics simulation were employed to study the impact of Cu­(II) binding on structures and dynamics of Aβ₄₂ monomer and dimers. It was shown that in the presence of Cu­(II) the β content of monomer is reduced substantially compared with the wild-type Aβ₄₂ suggesting that, in accord with experiments, metal ions facilitate formation of amorphous aggregates rather than amyloid fibrils with cross-β structures. In addition, one possible mechanism for amorphous assembly is that the Asp23–Lys28 salt bridge, which plays a crucial role in β sheet formation, becomes more flexible upon copper ion binding to the Aβ N-terminus. The simulation of dimers was conducted with the Cu­(II)/Aβ stoichiometric ratios of 1:1 and 1:2. For the 1:1 ratio Cu­(II) delays the Aβ dimerization process as observed in a number of experiments. The mechanism underlying this phenomenon is associated with slow formation of interchain salt bridges in dimer as well as with decreased hydrophobicity of monomer upon Cu-binding