Deconvoluting the Cu2+ Binding Modes of Full-length Prion Protein

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.

Dynamics of a truncated prion protein, PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

“…The N-terminal domain (residues 23-120) is highly disordered 15 and is notable for its ability to bind Cu 2þ ions. [16][17][18][19][20][21] The N-terminus contains a highly conserved octa-repeating sequence, PHGGGWGQ between residues 57 and 90. The flexible N-terminal tail of PrP C has shown some ordering at a lower pH of 4.…”

Conformational Properties of β-PrP

“…The disease-associated form of the protein, termed the scrapie form or PrP Sc , differs from the normal cellular form (PrP C ) through a conformational change, resulting in a significant increase in the ␤-sheet content and protease resistance of the protein (3,4). PrP C , in contrast, consists of a predominantly ␣-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5)(6)(7)(8)(9)(10)(11)(12). A single disulfide bond links two of the main ␣-helices and forms an integral part of the core of the structured domain (13,14).…”

Redox Control of Prion and Disease Pathogenesis

“…This phenomenon has been studied extensively by using recombinant PrP C , peptide fragments of PrP C , cell models, and mouse models (29,52,102,113,226). Collectively, these studies indicate that human PrP C binds five Cu 2þ ions under physiologic conditions.…”