Thermodynamic and spectroscopic investigation on the role of Met residues in CuII binding to the non-octarepeat site of the human prion protein

Abstract

Among the common features shared by neurodegenerative diseases there is the central role played by specific proteins or peptides which accumulate in neurons as insoluble plaques or tangles, containing abnormal amounts of redox-active metal ions, like copper and iron. In the case of transmissible spongiform encephalopathies (TSE), the involved protein is known as "prion protein" (PrP(C)) since "prions" (proteinaceous and infectious) are the agents which make TSE transmissible. It is widely accepted that PrP(C), in its wild-type form, can bind up to six Cu(II) ions, four of them in the so-called "octarepeat domain" and the others in the "fifth (non-octarepeat) binding-site". The latter domain contains two His residues, acting as anchoring sites for Cu(II) ions, and other potential binding residues, such as Lys and Met. While it is widely accepted that Lys residues do not take part in complex-formation, the role of methionines is still debated. In order to shed light on this issue, some peptides have been synthesized, either directly mimicking the sequence of the second half of the fifth binding site of human-PrP(C) (apo-form) or analogues where Met residues have been substituted by n-leucine. In addition, a series of short peptides, containing both His and Met residues in different relative positions, have been investigated, for the sake of comparison. Spectroscopic results, including NMR spectra of systems containing Ni(II) as a probe for the paramagnetic Cu(II) ion, agree on the exclusion of any direct interaction between the sulphur atom of Met residues and the Cu(II) ion already bound to His-imidazole side-chains. However, thermodynamic data show that Met-109 somewhat contributes to stability of complex species and this can be attributed to different electronic and steric effects.

Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

“…used different spectroscopy techniques, including EPR and NMR, to investigate copper coordination with Met 109 and Met 112 in several synthesized hPrP peptides. The results exclude any direct interaction between any Met residues and the copper ions that are already bound to His‐imidazole side chains …”

“…replaced Cu(II) with Ni(II) to study the coordination of six copper ions with PrP at physiological concentrations . In a recent study, Ni(II) was used instead of paramagnetic Cu(II) ions to reinvestigate copper interactions with Met residues in the non‐octarepeat site of the huPrP . Other metal ions, such as Pd(II) and Pt(II), were investigated as diamagnetic replacement ions such that proton NMR spectra can be recorded with 1 : 1 metal‐to‐protein stoichiometry …”

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

“…[77][78][79][80][81][82][83][84][85][86][87] Moreover, the hPrP91-127 region, which is next to the repeat domain, provides two further copper(II) binding sites, each associated with a histidine residue, His96 and His111, respectively. [67,70,72,73,75,76,[88][89][90][91][92][93][94][95][96] Although there is no general consensus, most of the results obtained so far indicate His111 as the preferential, if not exclusive, copper(II) binding site. [67,72,73,75,76,90,91,95,96] Our systematic studies carried out on small peptide fragments of PrP led to the same conclusions, and the high copper(II) binding affinities of the His96, [97] His111, [98] and even His187 [99] residues have unambiguously been demonstrated.…”

“…[67,70,72,73,75,76,[88][89][90][91][92][93][94][95][96] Although there is no general consensus, most of the results obtained so far indicate His111 as the preferential, if not exclusive, copper(II) binding site. [67,72,73,75,76,90,91,95,96] Our systematic studies carried out on small peptide fragments of PrP led to the same conclusions, and the high copper(II) binding affinities of the His96, [97] His111, [98] and even His187 [99] residues have unambiguously been demonstrated. A comparison of the thermodynamic stabilities of the histidinecontaining fragments revealed that the peptides that encompass the amino acid sequences outside the octarepeat domain are even more efficient chelators of copper(II) than the single octarepeat fragments.…”

Metal complexes of amino acids and peptides

“…Spectroscopic measurements on small model compounds and DFT calculations also supported these trends. 264 The possible inhibition of the aggregation of prion neuropeptides is common goal of many studies in this field. It was found that neither Zn(II) nor Ni(II) can substitute Cu(II) in the peptide complexes but can redistribute it among the available binding sites.…”