The Palladium(II) Complex of Aß4-16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids.

The Aß4-42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aß4-16 and Aß4-9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aß4-x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aß4-16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aß4-16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aß4-16, Pd(II)-Aß4-16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aß complexes.

Aß5-x Peptides: N-Terminal Truncation Yields Tunable Cu(II) Complexes.

The Aß5-x peptides (x = 38, 40, 42) are minor Aß species in normal brains but elevated upon the application of inhibitors of Aß processing enzymes. They are interesting from the point of view of coordination chemistry for the presence of an Arg-His metal binding sequence at their N-terminus capable of forming a 3-nitrogen (3N) three-coordinate chelate system. Similar sequences in other bioactive peptides were shown to bind Cu(II) ions in biological systems. Therefore, we investigated Cu(II) complex formation and reactivity of a series of truncated Aß5-x peptide models comprising the metal binding site: Aß5-9, Aß5-12, Aß5-12Y10F, and Aß5-16. Using CD and UV-vis spectroscopies and potentiometry, we found that all peptides coordinated the Cu(II) ion with substantial affinities higher than 3 × 1012 M-1 at pH 7.4 for Aß5-9 and Aß5-12. This affinity was elevated 3-fold in Aß5-16 by the formation of the internal macrochelate with the fourth coordination site occupied by the imidazole nitrogen of the His13 or His14 residue. A much higher boost of affinity could be achieved in Aß5-9 and Aß5-12 by adding appropriate amounts of the external imidazole ligand. The 3N Cu-Aß5-x complexes could be irreversibly reduced to Cu(I) at about -0.6 V vs Ag/AgCl and oxidized to Cu(III) at about 1.2 V vs Ag/AgCl. The internal or external imidazole coordination to the 3N core resulted in a slight destabilization of the Cu(I) state and stabilization of the Cu(III) state. Taken together these results indicate that Aß5-x peptides, which bind Cu(II) ions much more strongly than Aß1-x peptides and only slightly weaker than Aß4-x peptides could interfere with Cu(II) handling by these peptides, adding to copper dyshomeostasis in Alzheimer brains.

Metal-Peptide Complexes to Study Neurodegenerative Diseases.

Dishomeostasis of Cu(II) ions in the human body is connected with several serious diseases such as Alzheimer's disease or Wilson's disease. Therefore, a deep understanding of Cu(II)-binding properties to metal ions carriers, together with the knowledge about how they can interact with other copper-binding partners, e.g., amyloid-ß (Aß), is required to assess their relevance to the brain metal homeostasis. Ultraviolet-visible spectrometry (UV-Vis) and circular dichroism (CD) were used to study the coordination characteristics of Cu(II) with peptide containing the amino-terminal (H2N-Xaa-Yaa-His-) copper-binding (ATCUN) motif (Aß12-16-VHHQK-NH2) derived from Aß peptide.

Oligopeptides Generated by Neprilysin Degradation of ß-Amyloid Have the Highest Cu(II) Affinity in the Whole Aß Family.

The catabolism of ß-amyloid (Aß) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aß4-9 and a minor Aß12- x species. Alternative processing of the amyloid precursor protein by ß-secretase also generates the Aß11- 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 (Aß4-9), Glu-Val-His-His-Gln-Lys-am (Aß11-16), Val-His-His-Gln-Lys-am (Aß12-16), and pGlu-Val-His-His-Gln-Lys-am (pAß11-16), 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 (Aß4-9), 9.5 fM (Aß12-16), and 1.8 pM (Aß11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAß11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aß4-9 and Aß12-16 are the highest among the Cu(II) complexes of Aß 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 Aß4-9, a major Cu(II) binding Aß fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.

Interplay between Copper, Neprilysin, and N-Truncation of ß-Amyloid.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the ß-amyloid (Aß) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aß levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aß fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 µM),  while proteolysis of Aß yielded the soluble, Aß4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aß4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 µM) further suggests a mechanism for modulating NEP activity, Aß4-9 production, and Cu2+ homeostasis.

Interactions of α-Factor-1, a Yeast Pheromone, and Its Analogue with Copper(II) Ions and Low-Molecular-Weight Ligands Yield Very Stable Complexes.

α-Factor-1 (WHWLQLKPGQPMY), a peptidic pheromone of Saccharomyces cerevisiae yeast, contains a XHX type copper(II) binding N-terminal site. Using a soluble analogue, WHWSKNR-amide, we demonstrated that the W(1)H(2)W(3) site alone binds copper(II) with a Kd value of 0.18 pM at pH 7.4 and also binds imidazole (Im) in a ternary complex (Kd of 1 mM at pH 7.4). This interaction boosts the ability of the peptide to sequester copper(II) depending on the Im concentration up to a subfemtomolar range, not available for any oligopeptidic system studied before. Therefore, α-factor-1 and other XHX-type peptides are likely copper(II) carriers in biological systems.

Copper Exchange and Redox Activity of a Prototypical 8-Hydroxyquinoline: Implications for Therapeutic Chelation.

The N-truncated ß-amyloid (Aß) isoform Aß4-x is known to bind Cu(2+) via a redox-silent ATCUN motif with a conditional Kd = 30 fM at pH 7.4. This study characterizes the Cu(2+) interactions and redox activity of Aßx-16 (x = 1, 4) and 2-[(dimethylamino)-methyl-8-hydroxyquinoline, a terdentate 8-hydroxyquinoline (8HQ) with a conditional Kd(CuL) = 35 pM at pH 7.4. Metal transfer between Cu(Aß1-16), CuL, CuL2, and ternary CuL(NIm(Aß)) was rapid, while the corresponding equilibrium between L and Aß4-16 occurred slowly via a metastable CuL(NIm(Aß)) intermediate. Both CuL and CuL2 were redox-silent in the presence of ascorbate, but a CuL(NIm) complex can generate reactive oxygen species. Because the NIm(Aß) ligand will be readily exchangeable with NIm ligands of ubiquitous protein His side chains in vivo, this class of 8HQ ligand could transfer Cu(2+) from inert Cu(Aß4-x) to redox-active CuL(NIm). These findings have implications for the use of terdentate 8HQs as therapeutic chelators to treat neurodegenerative disease.

A Functional Role for Aß in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding.

Accumulation of the ß-amyloid (Aß) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aß1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aß4-42 peptide is a major Aß isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aß1-x peptides. By using Aß4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aß4-16 and Aß4-42 possess negligible redox activity. Combined with the predominance of Aß4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.

The impact of synthetic analogs of histidine on copper(II) and nickel(II) coordination properties to an albumin-like peptide. Possible leads towards new metallodrugs.

The purpose of our research was to obtain peptidomimetics possessing Cu(II) and Ni(II) binding properties, which would be useful for biomedical applications. In this context we used potentiometry, UV-VIS and CD spectroscopies to characterize the Cu(II) and Ni(II) binding properties of pentapeptide analogs of the N-terminal sequence of histatin 5. The peptides investigated had a general sequence DSXAK-am (am stands for C-terminal amide), with X including His and its three synthetic analogs, (4-thiazolyl)-L-alanine (1), (2-pyridyl)-L-alanine (2), and (pyrazol-1-yl)-L-alanine (3). The heterocyclic nitrogens present in these analogs were significantly more acidic than that of the His imidazole. We found that DSXAK-am peptides were able to bind Cu(II) and Ni(II) and form 4N complexes in a cooperative fashion, with similar affinities. These results indicate that acidic heterocyclic amino acids provide a viable alternative for histidine in peptidomimetics designed for metal ion binding.

Sequence-specific Cu(II)-dependent peptide bond hydrolysis: similarities and differences with the Ni(II)-dependent reaction.

Potentiometry and UV-vis and circular dichroism spectroscopies were applied to characterize Cu(II) coordination to the Ac-GASRHWKFL-NH2 peptide. Using HPLC and ESI-MS, we demonstrated that Cu(II) ions cause selective hydrolysis of the Ala-Ser peptide bond in this peptide and characterized the pH and temperature dependence of the reaction. We found that Cu(II)-dependent hydrolysis occurs solely in 4N complexes, in which the equatorial coordination positions of the Cu(II) ion are saturated by peptide donor atoms, namely, the pyridine-like nitrogen of the His imidazole ring and three preceding peptide bond nitrogens. Analysis of the reaction products led to the conclusion that Cu(II)-dependent hydrolysis proceeds according to the mechanism demonstrated previously for Ni(II) ions (Kopera, E.; Krezel, A.; Protas, A. M.; Belczyk, A.; Bonna, A.; Wyslouch-Cieszynska, A.; Poznanski, J.; Bal, W. Inorg. Chem. 2010, 49, 6636-6645). However, the pseudo-first-order reaction rate found for Cu(II) is, on average, 100 times lower than that for Ni(II) ions. The greater ability of Cu(II) ions to form 4N complexes at lower pH partially compensates for this difference in rates, resulting in similar hydrolytic activities for the two ions around pH 7.