Interaction of Copper(II) with the Prion Peptide Fragment HuPrP(76−114) Encompassing Four Histidyl Residues within and outside the Octarepeat Domain

Abstract

Complex formation processes between the 39-mer residue peptide fragment of human prion protein, HuPrP(76-114), and copper(II) ions have been studied by potentiometric, UV-vis, circular dichroism (CD), electron paramagnetic resonance, and electrospray ionization mass spectrometry methods. This peptide consists of 39 amino acid residues and contains two histidines (His77 and His85) belonging to the octarepeat domain and two histidines (His96 and His111) outside this domain. It was found that HuPrP(76-114) is able to bind 4 equiv of metal ions and all histidyl residues are independent, except nonequivalent metal binding sites in the oligonuclear species. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites below pH 5.5 in the form of various macrochelates. The macrochelation slightly suppresses, but cannot prevent, the deprotonation and metal ion coordination of amide functions, resulting in the formation of (N(im),N(-)), (N(im),N(-),N(-)), and (N(im),N(-),N(-),N(-))-coordinated copper(II) complexes in the pH range from 5.5 to 9. CD spectroscopy results gave clear evidence for the differences in the metal binding affinity of the histidyl sites according to the following order: His111 > His96 >> His77 approximately His85. Among the oligonuclear complexes, the formation of di- and tetranuclear species seems to be favored over the trinuclear ones, at pH values beyond the physiological ones. This phenomenon was not observed in the complex formation reactions of HuPrP(84-114), a peptide fragment containing only one histidyl residue from the octarepeat. As a consequence, the data support the existence of cooperativity in the metal binding ability of this peptide probably due to the presence of two octarepeat sequences of the dimeric octarepeat domain of HuPrP(76-114) at basic pH values.

Cross‐Talk Between the Octarepeat Domain and the Fifth Binding Site of Prion Protein Driven by the Interaction of Copper(II) with the N‐terminus

“…The compar- ison of the species-distribution diagram of copper(II) complexes with AcPrP(76-114) [102] with that pertinent to the metal complexes with AcPrP(84-114) [91] (Figure 3 b versus a) highlights that the formation of oligonuclear species decreases as the number of histidine residues increases. The low solubility of copper(II) complexes with AcPrP(60-114) did not allow a similar distribution diagram to be obtained, but the ESI-MS results are consistent with the potentiometric results discussed above.…”

“…This indicates that a further increase of the number of histidine Na adducts correspond to the same complex species but with Na ions in place of lysyl ammonium protons. [91,102] The numbers used for the identification of different species refer to p-r in L p H q Cu r (e.g., [LH 4 Cu] = 141).…”

“…Recently, we reported the speciation and structures of the complexes formed in the reaction of copper(II) with AcPrP(84-114) [91] (Ac = acetyl) and AcPrP . [102] These 31 and 39 amino acid peptides contain three or four histidine residues, respectively (His77, His85, His96, His111), which encompass histidyl residues inside and outside the octarepeat domain. We found that: 1) macrochelates formed below pH 5.5 are the primary and exclusive metal-binding mode of copper(II) complex species.…”

“…We investigated the complexation properties of the peptide by UV/Vis, circular dichroism (CD) and electrospray ionisation mass spectrometry methods and compared these results with those obtained for the copper(II) complexes with N-terminal peptide fragments previously studied in our laboratories. [91,102] In addition, limited proteolysis experiments together with mass spectrometric analysis of the peptide complexes were carried out to give further evidence of the coordination features for the copper(II)-AcPrP system. To complete our previous results, [91,102] we report that the formation of macrochelate species occurs in the physiological pH range and at low copper occupancy.…”

“…[91,102] In addition, limited proteolysis experiments together with mass spectrometric analysis of the peptide complexes were carried out to give further evidence of the coordination features for the copper(II)-AcPrP system. To complete our previous results, [91,102] we report that the formation of macrochelate species occurs in the physiological pH range and at low copper occupancy. At increased copper(II) ratios, however, amide-bound species form at the expense of macrochelate species.…”