The affinity of copper binding to the prion protein octarepeat domain : Evidence for negative cooperativity

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 PrPC, 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 (PrPSc) responsible for the TSEs. PrPC 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 of PrPC is unknown, but recent investigations focus on the ability of the octarepeat domain to take up copper (5, 7–11). PrPC protects against apoptosis (12) and radical-mediated oxidative damage (13, 14) and even stimulates nerve cell growth and development (15). Several copper-specific neuroprotective mechanisms have been proposed, including enzymatic function as a superoxide dismutase (SOD) (16), copper sequestration to inhibit deleterious oxidative chemistry (9, 17), and copper-dependent cellular signaling (5, 13). Figure 1 PrP possesses a flexible N-terminal domain and a globular C-terminal domain, made up of three R helices and two β strands, as shown above. Copper binds primarily in the octarepeat domain, composed of tandem repeats of the fundamental sequence ... To establish a molecular basis for the neuroprotective role of PrP, recent studies have focused on the elucidation of the chemical environment of the Cu2+-binding sites. Research from our laboratory demonstrates that, at pH 7.4, each HGGGW segment within the octarepeat domain binds one Cu2+ at saturation (18–20). In more recent work, we have shown that the octarepeat domain takes up Cu2+ in three distinct coordination modes, referred to as components 1, 2, and 3, controlled by the precise molar ratio of Cu2+ to protein, as shown in Figure 1 (17). These coordination modes are clearly discernible by a combination of electron paramagnetic resonance (EPR) techniques, including multifrequency and pulsed EPR. The component 3 coordination mode, which is observed at low copper occupancy, involves three to four octarepeats binding a single Cu2+ through the histidine imidazoles. At intermediate copper occupancy, the coordination mode switches to a mixture of components 2 and 1. In component 2, Cu2+ is bound to both the imidazole nitrogen and deprotonated amide nitrogen of histidine within a single octarepeat. A second imidazole nitrogen from a neighboring octarepeat is postulated to bind Cu2+ in the axial position (17). Component 1 arises at full copper occupancy and involves Cu2+ coordination to the HGGGW residues within each octarepeat through an imidazole nitrogen of histidine, deprotonated amide nitrogens from the two following glycines, and the amide carbonyl oxygen from the second glycine. The Trp participates through the formation of a hydrogen bond from the indole NH to an axially coordinated water (19). Determining the precise affinity for copper binding to PrP is essential for assessing the normal function of the protein. For an enzyme, one would expect high affinity, reflected through a low dissociation constant (Kd) (21), whereas buffering or sensing would require a lower affinity, with a Kd near the concentration of extracellular copper (22). Unfortunately, investigations into the dissociation constant and the mechanism of copper uptake reveal highly disparate outcomes depending upon the measurement technique and the particular PrP construct (8, 10, 21–25). Current estimates place Kd between 10−6 M (10, 22, 23) and 10−14 M (21, 24), an experimental uncertainty of 8 orders of magnitude. For example, mass spectrometry studies on PrP(57–91), encompassing all of the octarepeats, finds a dissociation constant of 0.2 μM for the first bound Cu2+, with the Kd increasing to 12 μM for the fourth Cu2+ (10). Similar micromolar affinities have been identified using amino acid competition studies, as detected by circular dichroism (CD) (23). Alternatively, binding curves determined from tryptophan fluorescence quenching from Cu2+ report a very high-affinity site with a Kd of 8 × 10−15 M (8 fM) in PrP(57–98) (24). Isothermal titration calorimetry (ITC) and competitive metal capture analysis (CMCA) also support the presence of a high-affinity Cu2+-binding site in the octarepeat region (21). Several reports suggest that the octarepeat domain takes up Cu2+ with positive cooperativity (8, 22, 25). For instance, equilibrium dialysis measurements performed on PrP(23–98) find a half-maximal binding at 5.9 μM and a Hill coefficient (n) of 3.4, indicating a very strong positive cooperativity through the micromolar range (8). Similar results were obtained from CD measurements of the d–d absorption band at 570 nm, arising from Cu2+ binding to PrP(58–91) (25). The CD signal increases in a sigmoidal fashion, consistent with positive cooperativity. In support of these studies, fluorescence quenching measurements suggest that binding occurs with a half-maximal value of 5.5 μM and a Hill coefficient of 2.4 (22). On the other hand, electrospray ionization (ESI) mass spectrometry performed on PrP(57–91) found a progressive decrease in the binding affinity as a function of the copper load, perhaps suggesting negative cooperativity (10). Positive cooperativity would exert a profound affect on the distribution of copper-occupied states. Specifically, concentrations of unbound and fully copper-occupied PrP should be dominant and substantially greater than partially copper-occupied proteins (26). This is certainly the case for oxygen binding to hemoglobin, which is characterized by a Hill coefficient of 2.8. This suppression of intermediate states is in contrast to the behavior recently observed by our laboratory in the elucidation of the three copper-dependent binding components in the octarepeat domain (17). EPR spectra suggest large populations of partially occupied intermediates, quite the opposite of positive cooperativity. The identification of distinct octarepeat binding modes in PrP motivates a re-evaluation of the affinity of the protein for copper. The individual modes may have widely differing dissociation constants and, if so, would help explain the wide discrepancy among previously published values. In addition, determining the affinity for each binding mode provides a quantitative assessment of cooperative copper uptake. Here, we use fluorescence quenching and EPR measurements on the octarepeat domain, along with a library of octarepeat-derived constructs and selective chelators in competition studies, to determine the affinity for the copper-binding components 1, 2, and 3. Next, we develop and apply an EPR approach for following the populations of the components as a function of copper occupancy to evaluate cooperative uptake. Using the population distribution, we then determine the Hill coefficient as a function of copper occupancy. In contrast to previous published measurements, we find that the affinity ranges from approximately 0.1 nM at low Cu2+ occupancy to 10 μM at high Cu2+ occupancy, with a Hill coefficient of less than 1.0 indicative of negative cooperativity. For select cases, we reconcile these findings with those that were previously interpreted as providing evidence of positive cooperativity.