Marek Łuczkowski

Copper Binding to the Neurotoxic Peptide PrP106–126: Thermodynamic and Structural Studies

The human prion protein fragment PrP106–126 is a highly fibrillogenic peptide, resistant to proteinases and toxic to neurons; it derives from the normal prion protein (PrPC), with which it can interact, thus inhibiting its superoxide dismutase‐like activity. The same properties are also shown by the abnormal isoform of the prion protein (PrPSc), and this similarity makes PrP106–126 an interesting model for the neurotoxic action of PrPSc. A role for copper in PrP106–126 aggregation and toxicity has recently been evidenced, and the interaction of terminal Lys, His and Met residues with the copper ion at neutral pH has been suggested. In order to shed more light on the complex‐formation equilibria of PrP106–126 with the copper ion, a thorough investigation has been carried out by means of several experimental techniques: potentiometry, solution calorimetry, VIS spectrophotometry, circular dichroism, EPR and NMR spectroscopy. A shorter and more soluble fragment—PrP106–113, which lacks the hydrophobic C‐terminal domain of PrP106–126 but contains all the potential donor groups—has also been considered for the sake of comparison. The involvement of terminal amino, imidazolic and amido nitrogens in complex formation has been confirmed, while no evidence was found for the interaction of side chains of Met and Lys residues with the copper ion. Solution structures for the main complexes are suggested.

Identification of a novel high affinity copper binding site in the APP(145–155) fragment of amyloid precursor protein

The copper(II) binding features of the APP(145-155) and APP(145-157) fragments of the amyloid precursor protein, Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-NH2 and Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 were studied by NMR spectroscopy and NMR findings were supported by UV-vis, CD and EPR spectra. Potentiometric measurements were performed only for the more soluble Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 peptide fragment. The following was shown: (i) the imidazole rings of all the three His residues are involved in metal coordination; (ii) metal binding induces ionisation of Leu-148 and His-149 amide nitrogens that complete the donor set to copper(II) in the species dominant at neutral pH; (iii) the unusual coordination scheme of the His-Xxx-His-Xxx-His consensus sequence justifies the high specificity for Cu(II) when compared to SOD-like or albumin-like peptides or even in amyloid Abeta fragments. The present findings may represent the key for interpreting the observed requirement of His residues conservation for the redox cycling between Cu(II) and Cu(I) by soluble APP.

Interactions of Cu2+ ions with chicken prion tandem repeats.

The potentiometric and spectroscopic (EPR, UV-Vis, CD) data have shown that the chicken prion hexa-repeat (Ac-His-Asn-Pro-Gly-Tyr-Pro-NH(2)) is a very specific ligand for Cu(2+) ions. The His imidazole is an anchoring binding site, then the adjacent amide nitrogen coordinates as a second donor. The presence of Pro at position 3 induces binding of phenolate oxygen as a third donor atom. The tridentate coordination dominates around physiological pH. Similar to human octapeptide fragments, chicken tandem repeats exhibit a cooperative effect in binding Cu(2+) ions, although chicken peptides are much less effective in metal ion coordination.

Is the monomeric prion octapeptide repeat PHGGGWGQ a specific ligand for Cu2+ ions?

Ac-PHGGGWGQ-NH2, an octarepeat peptide fragment of prion, is a relatively effective ligand for Cu2+ ions. At a pH of about 7.4 the major binding sites involve the imidazole nitrogen and two amide nitrogens of 3Gly and 4Gly giving a CuH−2L species. The stability of the complex formed is similar to other peptides having a similar type of coordination. The NMR spectra indicate that in CuH−2L the complex side chain of the Trp residue is located very close to the metal ion. The geometry around the Cu2+ ion seems to be slightly distorted from the tetragonal one. In strongly basic solution the coordination involves an additional amide nitrogen. In CuH−2L, CuH−3L and CuH−4L complexes the amide nitrogens involved in the metal ion binding are those placed towards the C-terminal from the His residue. The N-terminal of the unprotected octapeptide is very effective in binding the Cu2+ ion although at high pH the imidazole nitrogen may not be involved in metal ion binding.

Cu(II) ion coordination to SPARC: a model study on short peptide fragments

SPARC (secreted protein, acidic and rich in cysteine) is a glycoprotein of the extracellular matrix that mediates the cell-matrix interactions. It plays also a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. The human SPARC consists of three distinct modules. Module II is follistatin-like and its hydrolysis gives rise to a number of oligopeptides that can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. In order to completely understand the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the three SPARC122–126, SPARC121–126 and SPARC120–126 fragments, protected at both their amino and carboxylic ends, is reported. These peptides (Ac-HKLHL-NH2, Ac-GHKLHL-NH2 and Ac-KGHKLHL-NH2, respectively) constitute good models for the strong copper-binding site of the protein. The behaviour of the three ligands is very similar: complex formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles promotes amide ionization in the physiological pH range and this can explain SPARC binding to the Cu(II) ion.

Unusual gain in the coordination ability of vasopressin-like peptides towards Cu2+ ions by insertion of the highly hydrophobic side chain

Potentiometric and spectroscopic data show an unusual gain in the stability constants of Cu2+ complexes with vasopressin analogues having the highly hydrophobic naphthalene-alanine residue inserted in position three (Nal3). The naphthalene derivative is a much more powerful ligand for binding Cu2+ ions that the parent peptide. Theoretical calculations indicate the effective hydrophobic protection of the metal site by Tyr2 and Nal3 aromatic side-chains. The interaction of the guanidine moiety of Arg4 with naphthalene can also increase distinctly the stability of the respective 4N complex.

Specific binding of Cu2+ ions by a pentapeptide fragment present in the cysteine-rich region of amyloid precursor protein

The βA4 amyloid precursor protein fragment situated in the cysteine-rich region is a very effective binding site for Cu2+ ions due to the presence of three His residues in the His–Xaa–His–Yaa–His sequence.

Binding abilities of dehydropeptides towards Cu(II) and Ni(II) ions. Impact of Z–E isomerization on metal ion binding

The study on the binding ability of dehydro-tri- and tetrapeptides has shown that the alpha,beta-double bond has a critical effect on the peptide coordination to metal ions. It may affect the binding of the vicinal amide nitrogens by the electronic effect and stabilize the complex due to steric effects. The (Z) isomer is the most effective in stabilizing of the complexes formed. The presence of large side chain in the dehydroamino acid residue may also be critical for the coordination mode in the metallopeptide systems.

The possible role of Gly residues in the prion octarepeat region in the coordination of Cu2+ ions

Spectroscopic and potentiometric data have shown that insertion of tripeptides other than the Gly3 peptide fragment, Ala3 or Lys3, into the prion octarepeat region destabilizes the biologically relevant Cu2+ complex with the metal ion bound equatorially through the {Nimid,2N−} donor set. The other likely role of the high glycine content could be enforcement of the high flexibility of the N-terminal prion region resulting in the unstructured protein organization. However, the insertion of bulkier amino acid residues does not change the basic coordination mode at physiological pH which involves imidazole nitrogen and two amide nitrogen donors from the third and fourth residues.

Cu(II) ion coordination to the pentadecapeptide model of the SPARC copper-binding site

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein with many biological functions: it mediates the interactions between cells and the extracellular matrix, playing a role in angiogenesis, tumorigenesis, caractogenesis and wound healing. Proteolysis of SPARC gives rise to a number of oligopeptides which can regulate angiogenesis in vivo and the biological activity of which has been related to their association with endogenous or exogenous copper ion. Human SPARC consists of three distinct modules. Module II is follistatin-like and contains two copper binding sites, the strongest of which—the cationic region 2 (amino acids 114–130)—contains the sequence Gly–His–Lys. In order to shed more light on the biological role of metal complexes formed by SPARC and its fragments, more information is needed on their stoichiometry, stability and structure in solution. In the present paper a potentiometric and spectroscopic investigation on Cu(II) complexes with the SPARC114–128 fragment, protected at both its amino and carboxylic ends, is reported. This peptide (Ac–TLEGTKKGHKLHLDY–NH2) constitutes a good model to the strong copper-binding site of the protein. The whole experimental data suggest that complex-formation is started by the two His residues, subsequently involving up to three amido nitrogens, as pH increases. The coordination of the two histydyl imidazoles is able to promote amide ionisation in the physiological pH range and this could be the key to the SPARC affinity for Cu(II) ion.