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

Abstract

The prion protein (PrP) binds Cu 2+ in its N-terminal octarepeat domain, composed of four or more tandem PHGGGWGQ segments. Previous work from our laboratory demonstrates that copper interacts with the octarepeat domain through three distinct coordination modes at pH 7.4, depending upon the precise ratio of Cu 2+ to protein. Here, we apply both electron paramagnetic resonance (EPR) and fluorescence quenching to determine the copper affinity for each of these modes. At low copper occupancy, which favors multiple His coordination, the octarepeat domain binds Cu 2+ with a dissociation constant of 0.10 (±0.08) nM. In contrast, high copper occupancy, involving coordination through deprotonated amide nitrogens, exhibits a weaker affinity characterized by dissociation constants in the range of 7.0-12.0 μM. Decomposition of the EPR spectra reveals the proportions of all coordination species throughout the copper concentration range and identifies significant populations of intermediates, consistent with negative cooperativity. At most copper concentrations, the Hill coefficient is less than 1.0 and approximately 0.7 at half copper occupancy. These findings demonstrate that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude. Consideration of these findings, along with the demonstrated ability of the protein to quench copper redox activity at high occupancy, suggests that PrP may function to protect cells by scavenging excess copper.The prion protein (PrP) 1 is responsible for a novel class of infectious, neurodegenerative diseases collectively known as the transmissible spongiform encephalopathies (TSEs) (1-3). The TSEs include scrapie in sheep, mad cow disease (bovine spongiform encephalopathy, BSE), chronic wasting disease (CWD) in deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans. The normal cellular form of the prion protein, referred to as PrP C , is found in a wide range of tissues of all mammalian and avian species. A misfolding event converts the protein to the infectious, β-sheet-rich scrapie form (PrP Sc ) responsible for the TSEs.PrP C is a GPI-anchored glycoprotein expressed in abundance on the surface of neurons, predominantly on presynaptic membranes (4,5). The mature form of PrP, consisting of residues 23-231 in hamster, has a globular C-terminal domain and a largely unstructured N-terminal domain (6) (Figure 1). The C-terminal domain (residues 125-231) contains three α helices, two of which are stabilized by a single interhelical disulfide bond, and a small antiparallel β sheet. The flexible N-terminal domain is glycine-rich and highly flexible. Within the flexible N-terminal region of PrP is the octarepeat domain, composed of four or five highly conserved, contiguous repeats of the eight-residue sequence PHGGGWGQ. The physiological function

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

“…8c. Closer inspection of the hPrP-(58 -91) spectra indicates the first Cu 2ϩ ion binding to the octarepeats produce spectra typical of a multiple His complex, complex I, described previously (44). After addition of further Upon the addition of just 0.2 mol eq of HIMDA, the hyperfine A ʈ splitting reduces in magnitude and overall, the g ʈ signals shift to higher field strengths.…”

“…An important observation is to note that Cu 2ϩ added to full-length PrP produces a significant visible CD signal even at substoichiometric amounts of Cu 2ϩ . Coordination of Cu 2ϩ to multiple His has previously been observed (complex I) (43,44). This type of complex with no main chain coordination from amides, will be CD silent in the visible region (43,45 (20,43,44,48,49).…”

“…1. Affinity for this binding mode has been placed at a K d of 1-10 M at physiological pH (41,43,44). Recent studies have shown that at substoichiometric levels, Cu 2ϩ will bind to multiple His residues with a higher affinity, K d in the 0.1-3 nM range (43,44) (complex I), also shown in Fig.…”

“…Subsequent Cu 2ϩ ions bind to individual His residues within the octarepeats (complex II). Coordination of Cu 2ϩ to multiple histidines in the octarepeats (complex I) (43,44), is not ruled out. However, this complex is CD silent and is therefore clear complex I does not make a signifi- cant contribution to the initial 2 mol eq of Cu 2ϩ loaded onto PrP, as the intensity of the signal at 580 nm is apparent even at 0.…”

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).…”