Unraveling the Cu²⁺ Binding Sites in the C-Terminal Domain of the Murine Prion Protein:  A Pulse EPR and ENDOR Study

Abstract

The mammalian prion protein (PrPC) is a cell surface protein consisting of a flexibly disordered N-terminal segment (residues 23−120) and a structured C-terminal domain (residues 121−231). PrPC is supposed to bind Cu2+ in vivo, and several studies have recently focused on the ability of this protein to bind divalent cations. In a previous continuous wave electron paramagnetic resonance (CW EPR) study, we showed that Cu(II) binds both to the N- and C-terminal parts of PrPC. Here we present a pulse EPR and electron nuclear double resonance (ENDOR) study of the three different Cu(II) binding sites observed in the structured, C-terminal part of the murine prion protein, mPrP(121−231). It was found that the three complexes are distinguished by a different number of nitrogen atoms directly involved in the Cu(II) ligation. For one of the Cu(II) binding sites that is observed at low pH (3−6), no directly coupled nitrogens could be observed. For a second type of Cu(II) complex, observed at pH 3−8, Davies-ENDOR and hyperfine sublevel correlation (HYSCORE) spectroscopy revealed that histidine is one of the binding ligands. Furthermore, the presence of a nonexchangeable proton close to a copper ion could be demonstrated in a sample containing mainly the second Cu(II) complex. For the third mode of Cu(II) complexation, which can be detected at pH 7−8, Davies-ENDOR spectra indicate that more than one nitrogen atom is directly bound to the copper ion. The observed EPR parameters suggest the involvement of backbone nitrogens in this copper(II) complex.

Comparative Analysis of the Human and Chicken Prion Protein Copper Binding Regions at pH 6.5

“…Our results evolved at a pH of 6.5, which is close to conditions in the endocytic compartments of neuronal cells where the Cu(II) is potentially released, closing the gap between the detailed structural models determined at pH 7.4 and the studies performed at pH 6.0 (48). In addition, the reduced ability of copper binding suggested for the chicken PrP at high Cu(II) concentrations in the environments of membrane rafts is highly indicative of different physiological functions of human and chicken prion proteins.…”

Copper and the Prion Protein: Methods, Structures, Function, and Disease

“…As indicated by the hyperfine splittings, both g ‖ and A ‖ shift with increasing pH. This is attributable to a progressive transition from 4O coordination at low pH to 4N coordination at high pH (52,53). There are several archetypal copper centers readily identified by their EPR spectra.…”

Micellar environments induce structuring of the N‐terminal tail of the prion protein

“…18,19 -21 Moreover, downstream of this repeat an additional Cu 2ϩ binding site was identified 22,23 which involves the His residue 96. 24 Copper complexes of PrP c and its N-terminal fragments as well as of related synthetic peptides have been extensively investigated by various spectroscopic techniques, 18 -29 cyclic voltamperometry, 19,21,30 and potentiometric measurements 31 as well as by NMR 31 and x-ray analysis. 32 A consensus picture emerged from these studies about the stoichiometry of the metal complexes (Cu 2ϩ /octapeptide and Cu 2ϩ / (90 -113)-peptide ratios of 1:1 at pH Ն 7) and binding affinities (K d values in the low micromolar range).…”