Electron Paramagnetic Resonance Evidence for Binding of Cu2+ to the C-terminal Domain of the Murine Prion Protein

Abstract

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.

Mapping Cu(II) Binding Sites in Prion Proteins by Diethyl Pyrocarbonate Modification and Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometric Footprinting

“…The only apparently discordant determination concerns experiments using EPR analysis. Although precise stoichiometries were not emphasized, spectra for two coordination geometries not seen for N-terminal fragments were indicative of two varieties of Cu(II) binding sites specific within PrP121-231 (50). One type of site was compatible with an oxygen-dependent ligation, perhaps via aspartic or glutamic acid residues and was seen at pH values Ͻ7.0 (and therefore potentially …”

High Affinity Binding between Copper and Full-length Prion Protein Identified by Two Different Techniques

“…Although four of these sites were designated as belonging to the octameric repeat region, the location of the so-called fifth site has been difficult to identify. There have been three suggestions, each associated with a histidine at either amino acid residue 96 (9), 111 (12), or 187 (13,14). However, the majority of reports suggest that, if there is a fifth site, it is also in the N terminus.…”

Inter- and Intra-octarepeat Cu(II) Site Geometries in the Prion Protein

“…These residues are located within the tandemly repeated PHGGGWGQ sequence known as the octarepeat and at the flexible hinge that connects the N-terminal and C-terminal domains (16 -20). In addition to these, other minor Cu(II)-binding sites in the C-terminal globular domain have been reported (19,21,22). Studies with PrP C models, as synthetic peptides and recombinant forms, have shown binding stoichiometries of up to one Cu(II) per octarepeat motive in a process in which the interaction among cation sites, measured as positive cooperativity, appears when the number of sites is higher than two (15,18,(22)(23)(24).…”