No abstract

Sc , is a proteinaceous infectious particle, devoid of nucleic acids, which is responsible for bovine spongiform encephalopathy in cattle and a number of prion diseases in humans, including Creutzfeldt Jakob Disease (1-3). Normal cellular prion protein (PrP C ) is typically 209 residues long, attached to the cell surface by a glycosylphosphatidylinositol anchor. The C-terminal domain between residues 126 and 231 is mainly ␣-helical (4). In contrast, in the absence of copper ions, the N-terminal domain between residues 23 and 125 is unstructured (5) and exhibits a high degree of main chain flexibility (6). It is this natively unfolded domain that includes octarepeat sequences that bind a number of Cu 2ϩ ions (7-9). Metal imbalance is a feature of prion disease (10) and when isolated from diseased brain, PrP Sc has been found to be occupied with metal (11). Metal binding to the prion protein is altered in human prion disease (12), with levels of cellular copper affected by scrapie infection (13) and the ability for disease progression in infected mice to be slowed with the use of copper-specific chelation therapy (14). Copper-catalyzed redox damage of PrP (15) is linked to prion disease (16, 17), although, the copper binding octarepeat region is not required for prion infectivity and propagation (18). Cu 2ϩ binding has also been noted outside the octarepeats, in the so called 5th site (8, 19 -23). This region is an amyloidogenic, neurotoxic segment of PrP that is essential for prion replication (18, 24 -29). Interestingly, the presence, or absence, of Cu 2ϩ ions may also confer different strains of prion disease (11, 30). PrP C is concentrated at presynaptic membranes in the central nervous system, where Cu 2ϩ is also highly localized (32). The ability of PrP C to bind Cu 2ϩ in vivo and in vitro infers PrP may have a physiological role in copper homeostasis (7, 9). Copper has been shown to promote the endocytosis of PrP C (33, 34), but PrP expression levels do not seem to affect copper delivery (35,36). Copper-induced cleavage of the PrP C main chain has also been reported (37,38) (22), and mass spectrometry (30).There have been numerous Cu 2ϩ binding studies of fragments of PrP centered on two regions within the unstructured N-terminal domain of PrP. These studies include the octarepeat region, residues 58 -91, and a second Cu 2ϩ binding region between the octarepeats and the C-terminal structured domain, between residues 90 and 126. Mammalian PrPs contain a repeating motif of 8 amino acids, typically, four octarepeats between residues 60 and 91 with each repeat containing a histidine residue. It is this unstructured, highly conserved * This work was supported in part by Biotechnology and Biological Sciences Research Council Project grants. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The prion protein (PrP) is a Cu2+-binding cell-surface glycoprotein. Using various PrP fragments and spectroscopic techniques, we show that two Cu2+ ions bind to a region between residues 90 and 126. This region incorporates the neurotoxic portion of PrP, vital for prion propagation in transmissible spongiform encephalopathies. Pentapeptides PrP-(92-96) and PrP-(107-111) represent the minimum motif for Cu2+ binding to the PrP-(90-126) fragment. Consequently, we were surprised that the appearance of the visible CD spectra for two fragments of PrP, residues 90-126 and 91-115, are very different. We have shown that these differences do not arise from a change in the co-ordination geometry within the two fragments; rather, there is a change in the relative preference for the two binding sites centred at His111 and His96. These preferences are metal-, pH- and chain-length dependent. CD indicates that Cu2+ initially fills the site at His111 within the PrP-(90-126) fragment. The pH-dependence of the Cu2+ co-ordination is studied using EPR, visible CD and absorption spectroscopy. We present evidence that, at low pH (5.5) and sub-stoichiometric amounts of Cu2+, a multiple histidine complex forms, but, at neutral pH, Cu2+ binds to individual histidine residues. We have shown that changes in pH and levels of extracellular Cu2+ will affect the co-ordination mode, which has implications for the affinity, folding and redox properties of Cu-PrP.

A natively unfolded region of the prion protein, H NMR suggests that the coordination geometry for the two binding sites is very similar. However, the visible CD spectra of the two sites are very different, producing almost mirror image spectra. We have used a series of analogues of the pentapeptides containing His 96 and His 111 to rationalise these differences in the visible CD spectra. Using simple histidine-containing tri-peptides we have formulated a set of empirical rules that can predict the appearance of Cu 2+ visible CD spectra involving histidine and amide main-chain coordination.

PrP (prion-related protein) is a cell-surface Cu(2+)-binding glycoprotein which, when misfolded, is responsible for a number of transmissible spongiform encephalopathies. The co-ordination geometry, stoichiometry and affinity of Cu(2+) for PrP are the subject of much debate. In the present paper, we review the recent progress we have made in these areas. As many as six Cu(2+) ions bind to PrP with submicromolar affinity. Initially, two Cu(2+) ions bind to full-length PrP in the amyloidogenic region, between the octarepeats and the structured domain, at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Competitive chelators have been used to determine the affinity of the first molar equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+)-binding sites support the suggestion that PrP could act as an antioxidant by binding potentially harmful Cu(2+) ions and sacrificially quenching of free radicals generated as a result of copper redox cycling. Finally, the effect of Cu(2+) on the prion structure and misassembly into oligomers and fibres is discussed.