Copper Coordination in the Full-Length, Recombinant Prion Protein

Abstract

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.Prion diseases are fatal neurodegenerative disorders of both humans and animals (1). The causative agent is an isoform of a normal, host-encoded membrane glycoprotein called the prion protein (PrP). 1 The normal cellular isoform (PrP C ) is the precursor to the pathogenic, protease-resistant isoform termed PrP Sc , which is responsible for homologous pathologies within its individual hosts. With rare but notable exceptions, prion diseases respect the species barrier (2,3). Among these exceptions is the transmission of disease from scrapie-infected † This work was supported by NIH Grants GM 65790 (G.L.M.), GM 60609 (G.J.G.), GM 40168 (J.P.), AG02132 and AG10770 (S.B.P.).

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

“…Clearly, the results reported here require not only confirmation in full-length PrP but also an assessment of the influence of non-octarepeat coordination sites (20,28) and other features that may affect Cu 2+ uptake. With regard to octarepeat coordination, however, our previously published experiments on full-length PrP Since the discovery of the physiological connection of PrP to copper, there have been intensive efforts to determine the precise Cu 2+ affinity (8,10,(21)(22)(23)(24)(25).…”

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

“…These models, which were elaborated considering the substoichiometric complexations of Cu(II) per octarepeat, differ in the final assembly, being either helical or planar. The models of the second group, which corresponds to full site occupancy, are formed in the crystal structure of Cu(II) in complex with the minimal site HGGGW and are then considered to be applicable to the fully saturated SHaPrP-(23-231) (17,19). In these fully saturated forms, the Cu(II) geometry is one of penta-coordination with the nearly equatorial ligation to the ␦1 nitrogen of the His, the deprotonated amide nitrogens of the two sequential Gly residues, the amide carbonyl oxygen of the second Gly * This work was supported in part by Ministerio de Ciencia y Tecnología of Spain (MCYT) Grants BIO2000-1664, BIO-2000-0175-P4, and BIO2003-00285, and a NeuroPharma S.A.-CSIC contract (all to M. G.).…”

“…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).…”

Comparative bioinformatics analysis of prion proteins isolated from reptile, rodent, ruminant, and human species

“…PrP binds copper with low-micromolar affinity and in a pH-sensitive manner via N-terminal histidine-containing octapeptide repeats (Jackson et al, 2001;Kramer et al, 2001;Burns et al, 2002). Binding of Cu ++ to the C-terminal portion of PrP in vivo seems unlikely, but it has been suggested by experiments using amino-terminal truncations of the protein (Burns et al, 2003).…”