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Originally published In Press as doi:10.1074/jbc.M709820200 on March 10, 2008

J. Biol. Chem., Vol. 283, Issue 19, 12831-12839, May 9, 2008
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Manganese Binding to the Prion Protein*

Marcus W. Brazier{ddagger}, Paul Davies{ddagger}, Esmie Player{ddagger}, Frank Marken§, John H. Viles, and David R. Brown{ddagger}1

From the Departments of {ddagger}Biology and Biochemistry and §Chemistry, University of Bath, Bath BA2 7AY, United Kingdom and School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom

Received for publication, December 3, 2007 , and in revised form, February 27, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
There is considerable evidence that the prion protein binds copper. However, there have also been suggestions that prion protein (PrP) binds manganese. We used isothermal titration calorimetry to identify the manganese binding sites in wild-type mouse PrP. The protein showed two manganese binding sites with affinities that would bind manganese at concentrations of 63 and 200 µM at pH 5.5. This indicates that PrP binds manganese with affinity similar to other known manganese-binding proteins. Further study indicated that the main manganese binding site is associated with His-95 in the so-called "fifth site" normally associated with copper binding. Additionally, it was shown that occupancy by copper does not prevent manganese binding. Under these conditions, manganese binding resulted in an altered conformation of PrP, displacement of copper, and altered redox chemistry of the metal-protein complex. Cyclic voltammetric measurements suggested a complex redox chemistry involving manganese bound to PrP, whereas copper-bound PrP was able to undergo fully reversible electron cycling. Additionally, manganese binding to PrP converted it to a form able to catalyze aggregation of metal-free PrP. These results further support the notion that manganese binding could cause a conformation change in PrP and trigger changes in the protein similar to those associated with prion disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cellular prion protein (PrPc)2 is a metal-binding glycoprotein expressed on the plasma membrane of a variety of cell types (1). In particular, PrPc is highly expressed at neuronal synapses (2). The function of the protein is still debated, but data have suggested a number of possible different roles, including an antioxidant, a copper transport protein, and involvement in cell adhesion or cell signaling (3–6). Although the role of the protein in normal cell activity is still being defined, it is widely agreed that conversion of PrPc to an abnormal isoform (PrPSc) plays a central role in the disease process of a family of disorders termed prion diseases or transmissible spongiform encephalopathies (1). These diseases include sporadic forms such as scrapie and Creutzfeldt-Jakob disease, inherited forms such as fatal familial insomnia and transmitted forms such as bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease.

The majority of data clearly point to PrPc being a cuproprotein (7–13). In addition, various cellular activities have been suggested resulting from the interaction of copper and PrPc (3, 4, 14, 15). As well as possible beneficial effects of copper binding, other data suggest that inappropriate interactions between copper and PrP result in the aggregation of the protein and increased proteinase K resistance (16, 17). Other studies have shown that copper can enhance the infectivity of prions and that copper chelators extend the incubation period of the disease (18, 19). This picture is further complicated by the fact that copper can also prevent prion fibrils from forming (20), and increased copper in the diet can delay the onset of prion disease symptoms (21).

Other metals have been suggested to bind to PrPc. These include manganese, zinc, and nickel (8, 11, 22–25). The binding of manganese to PrP potentially results in the conversion of the protein to an abnormal isoform with properties reminiscent of PrPSc (8, 26). In particular, manganese-bound PrP shows greater protease resistance (27), increased β-sheet content, the ability to aggregate (28), and the ability to seed polymerization of further prion protein (26). These effects are observed in recombinant protein and protein expressed in cells (29). It has been suggested that substitution of copper with manganese could bring about changes in PrP, initiating prion disease (8). Despite geochemical evidence that high environmental manganese coincides with some clustering of prion disease cases (30) and the cellular and biochemical effect of manganese on PrP, there is currently no evidence that exposure to increased levels of manganese is anything other than a potential risk factor for prion diseases.

Despite the interest in manganese binding to PrP, a thorough analysis of the interaction of the metal with the protein has not appeared. In this study we examined the affinity of manganese for PrP and demonstrated that manganese can bind to PrP even while copper is still bound and induce a conformational change. The use of cyclic voltammetry indicated that three copper redox-active centers are displaced in manganese-charged PrP and that the metal-protein complex has an estimated reversible potential of +0.08 V. We showed that the main manganese binding center of the protein is associated with a histidine a position 95 in the mouse PrP sequence with an affinity equivalent to that of other known manganese-binding proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Unless stated, all reagents were purchased from Sigma.

Protein Purification—Recombinant mouse prion proteins were prepared as described previously (13). Briefly bacterial expression was used to generate recombinant protein. Bacteria were transformed with a plasmid (pET) containing the open reading frame of wild-type mouse PrP (amino acid residues 23–231) or mutants of this construct. Protein expression was induced with 1 mM isopropyl 1-thio-β-D-galactopyranoside, and inclusion bodies were isolated from the bacteria with standard techniques. The inclusion bodies were solubilized in a buffer containing 8 M urea. Recombinant PrP was purified using immobilized metal affinity chromatography. The column was charged with copper, and the protein bound to the column was eluted with 300 mM imidazole in 8 M urea. All proteins were generated tag-free. 0.5 mM EDTA was added to the protein to chelate any metals present. All subsequent steps used double deionized water treated with Chelex resin to remove residual metal ions. The denatured protein was refolded by a 10-fold dilution of the urea in deionized water, the protein was concentrated by ultrafiltration, and two rounds of dialysis were carried out to remove residual urea, imidazole, and EDTA. Protein concentrations were measured using theoretical extinction coefficients at 280 nm (ExPASy ProtParam tool) and confirmed by BCA assay. Protein purity was checked using polyacrylamide gel electrophoresis under denaturing conditions and staining with Coomassie Brilliant Blue.

For some experiments recombinant proteins were charged with metal (either MnSO4 or CuSO4) during the refolding process. This results in saturation of the available metal binding sites with the metal. The method was as described previously (8), and the metal occupancy was confirmed with mass spectroscopy. For some experiments PrP was treated with diethyl pyrocarbonate (DEPC) to block potential metal binding sites. As PrP contains seven histidine molecules, DEPC was incubated with PrP at a 10 M excess (in 50 mM Na2PO4, pH 6.8) to ensure that all histidine molecules were modified. Modification of histidine was monitored as increased absorbance at 245 nm. Once the reaction was complete excess DEPC was removed by dialysis.

The mutant proteins used in this study were generated as described previously (13, 31). The mutant proteins used in this study include a deletion of N-terminal residues 23–89 (PrP90–231), deletion of residues 23–112 (PrP113–231), deletion of the octameric repeat region 51–89, and three mutants with alanine substituted for histidine at amino acid residues 95, 110, or both (H95A, H110A, and H95A/H110A). In addition two peptides were used for these experiments. The first was a 32-amino acid residue peptide based on the octameric repeat sequence (residues 51–89) as described previously (8) termed octarepeat (PHGGGWGQPHGGSWGQPHGGSWGQPHGGGWGQ). The second was a peptide with the sequence equivalent to amino acid residues 90–120 of the mouse PrP sequence (GGGTHNQWNKPSKPKTNLKHVAGAAAAGAVV). Both peptides were synthesized with modified N- and C-terminal residues to ensure no nonspecific metal interactions with terminal groups as described previously (32).

Isothermal Titration Calorimetry—Isothermal titration calorimetry (ITC) was carried out as described previously (13). All measurements were made on a MicroCal VP-Isothermal Titration Calorimeter instrument. Briefly a time course of injections of metal to PrP was made in an enclosed reaction cell maintained at a constant temperature. The instrument measured the heat generated or absorbed as a ligand-macromolecule reaction occurred. A binding isotherm was fitted to the data, expressed in terms of heat change per mole of metal against metal to PrP molar ratio. From the binding isotherm values for the reaction stoichiometry and association constants (Ka) were obtained.

All solutions were filtered through a 0.22-µm filter and degassed prior to use. Typically an initial injection of 2 µl of metal solution was followed by a further 29 injections of 4 µl of metal solution into the protein in the sample cell stirred at 300 rpm. Injections were separated by 120 s to allow equilibration, and sample temperature was maintained at 25 °C. All experiments were repeated at least three times. Data were analyzed with the Origin 5.0 software package from MicroCal. A baseline correction was applied to each experiment by subtraction of data from a series of injections of metal solution into a buffer blank correlating to the heat of dilution of the metal solution. After subtraction of the blank data a nonlinear least squares method was used to minimize {chi}2 values and obtain best fit parameters for the association constants. In all cases best fit parameters were obtained from the sequential binding site model whereby the user defines the number of binding sites to be fitted in a sequential manner.

Circular Dichroism Spectroscopy—For analysis of protein samples by circular dichroism (CD) spectroscopy the samples were diluted to 0.1 mg/ml. The protein concentration was determined for the samples by first assessing the concentration of a metal-free PrP sample using a scan of the UV spectra with a Carey spectrophotometer (Varian) using an extinction coefficient of 63,495 M–1 cm–1. As metals can alter the UV absorbance of protein, a Bradford assay was used to assure that the metal-binding PrP samples were at the same concentration as apoPrP. CD spectra for apoPrP and metal-charged PrP were obtained using an Applied Photophysics Chirascan spectropolarimeter as described previously (31). Protein concentration and path length were used to equate the measured spectra and plotted in the form of molar ellipticity between 185 and 260 nm.

Cyclic Voltammetry—Voltammetric measurements were conducted with a µ-Autolab III potentiostat system (Eco Chemie, Utrecht, The Netherlands) in a conventional three-electrode electrochemical cell. Experiments were performed in staircase voltammetry mode with platinum gauze counter and saturated calomel reference electrode (REF401, Radiometer Analytical). The working electrodes used were 5-mm-diameter edge plane pyrolytic graphite (Pyrocarbon, Le Carbone, East Sussex, UK) or 3-mm-diameter boron-doped diamond (Windsor Scientific, Slough Berkshire, UK). Electrodes were polished on fresh cloths (Buehler, Coventry, UK) with alumina (1 µm; Buehler) as a polishing aid. After the final polish on a clean cloth, electrodes were rinsed with demineralized water. Aqueous solutions were thoroughly deaerated with nitrogen (BOC Industrial, Manchester, UK) prior to recording data. All measurements were undertaken at 25 ± 2 °C. For voltammetric measurements an aqueous buffer solution (5 mM MES at pH 7) was thoroughly deaerated with nitrogen. The working electrode was polished, and the background current was recorded in the absence of protein. Next the working electrode was immersed into the protein solution (containing 20 µM wild-type mouse PrP (WT-PrP) in MilliQ filtered water, pH 7) and after 60 s removed and rinsed with MilliQ filtered water, pH 7. The resulting protein-modified electrode was reimmersed into the pure buffer solution in the measurement cell, and cyclic voltammograms were recorded. Protein adhesion to the edge plane pyrolytic graphite and to the boron-doped diamond electrode surfaces was excellent, and stable signals were obtained for many potential cycles. Redox potentials were also determined with reference to the saturated calomel electrode (reference electrode).


Figure 1
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  		FIGURE 1.
ITC analysis of manganese binding to PrP. A, WT-PrP was placed in the ITC calorimeter, and MnSO4 was titrated into the solution at pH 5.5. Shown is a typical trace indicating the raw energy change per injection (upper panel) and the net energy change for the titrations in relation to the moles of manganese injected. Manganese binding produces a strong isotherm as indicated by the curve. B, WT-PrP was modified with DEPC. Experiments were repeated with the same conditions as for A. In this case there was little evidence for metal binding, and no isotherm was obtained. C, ITC experiments with unmodified WT-PrP were carried out at a variety of pH values to determine the pH sensitivity of the manganese binding sites. Experiments were repeated three times. The highest affinity values were seen at pH 5.5. As the values are on a log scale, no error is shown. However standard error values were less than 5% of the mean values. {blacksquare}, high affinity site; {triangleup}, low affinity site.

 
Aggregation Studies—Recombinant PrP was generated as described above and either refolded to bind manganese or refolded to bind copper. Some copper-refolded protein was exposed to manganese as described for ITC experiments. The basis for the experiment was the ability of the metal-charged protein to cause aggregation of monomeric, metal-free PrP as determined by a change in absorbance at 325 nm. Metal-free WT-PrP was prepared at 1 mg/ml in 1 mM MES, pH 6.5. 1 ml of this solution was placed into a cuvette, and 0.1 mg/ml seeding PrP (with bound metals) was added in 50 µl of 1 mM MES, pH 6.5. All measurements were performed using a Cary 100Bio UV-visible spectrophotometer (Varian) at 325 nm using a quartz cuvette of 5-mm path length. Polymerization was observed as an increase in solution turbidity. The resultant scattering of UV light at 325 nm resulted in an increased absorbance measurement. Measurements were taken at 1–10-min intervals until no further change in absorbance was measured in all samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Manganese Affinity for PrP and pH Dependence—We used ITC to study manganese binding to mouse PrP. WT-PrP was generated by expression in bacteria and isolated using an immobilized metal affinity chromatography technique. This protein consisted of the full-length mouse PrP lacking the signal and glycosylphosphatidylinositol signal peptides (PrP23–231). The purified protein was tag-free and dialyzed after treatment with EDTA to remove any trace metal contamination. Manganese was titrated into PrP at a variety of pH values to determine the affinity within the pH range of 4.5–8. ITC experiments showed strong isotherms for the binding of manganese (Fig. 1). Using a data fitting program, two sequential bindings sites were determined, and the affinity for each site was calculated. Two sites were identified at all pH values tested. Fig. 1 shows that the highest affinity of manganese for WT-PrP was observed at pH 5.5. At this pH the calculated dissociation constants for the two sites were 63 and 200 µM. These values lie within the expected range for manganese affinity. At pH 7.5 the dissociation constant values rose to 630 µM and ~10 mM. At this pH it is unlikely that the second site would be occupied. Further experiments were carried out at pH 5.5.

Previous studies have suggested that manganese binds to PrP in association with histidine residues. Proteins can be specifically modified by reaction of DEPC with the imidazole groups of histidines, blocking the ability to bind metals. To assess whether histidines were important to manganese binding, PrP was modified with DEPC, and ITC experiments were repeated at pH 5.5. As shown in Fig. 1B, no manganese binding to PrP was observed. This suggests that histidine residues play a central role in manganese binding to PrP and further supports the notion that manganese is a ligand for PrP.

Identification of Manganese Binding Sites—To assess the location of the two manganese binding sites, mutant forms of PrP were also used in ITC experiments. Table 1 shows the results of the ITC experiments and includes the affinity values calculated. Only one of the mutants showed no binding of manganese. The mutant PrP113–231 lacks the N terminus of the protein. This indicates that both sites lie within the N terminus. Deletion of the N terminus up to residue 90 (i.e. PrP90–231) resulted in the loss of the low affinity binding site suggesting that the high affinity site lies between residues 90 and 112. Analysis of a mutant lacking only the octameric repeat region confirmed that the low affinity site lies within the octameric repeat. To confirm this we used two synthetic peptides corresponding to these two regions. The peptide 90–120 bound manganese with high affinity, an order of magnitude higher than for the wild-type protein PrP23–231. In comparison the affinity for the octarepeat peptide was very low, confirming work from previous studies (33).


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  		TABLE 1
Affinity of manganese for PrP mutants

Shown are the log of the affinity values (M–1) determined from ITC experiments in which MnSO4 was titrated into a solution of PrP protein at pH 5.5 buffered with 5 mM MES. ND, not detected. Site 1 is the high affinity site associated with the fifth site; site 2 is that associated with the octameric repeat region. Experiments were repeated three times.

 
As studies with DEPC suggested that manganese binds to histidine in the protein and two histidines form the so-called "fifth site" for copper binding, we examined mutants lacking either or both of these histidines. The double mutant with alanine substituted at His-95 and His-110 lacked the high affinity site. This confirms that this site is the high affinity manganese binding site in PrP. Substitution of His-95 alone resulted in loss of the high affinity site, whereas substitution of His-110 did not (Table 1). This implies that His-95 is central to the high affinity manganese binding site of PrP.

Metal Binding to Manganese-saturated PrP—To assess whether copper and manganese bind to equivalent sites in the protein we carried out experiments with manganese-saturated PrP. Following purification of PrP in the presence of 8 M urea, the protein was refolded to its native conformation in the presence of manganese. This results in occupation of all available manganese binding sites (8). Protein purified in this way was subjected to ITC experiments with manganese. Manganese binding to manganese-saturated PrP resulted in one binding event (Fig. 2) with very low affinity (Table 2). Under the same conditions manganese-saturated H95A/H110A also showed binding at one site with the same affinity. Therefore this low affinity binding site (if physiologically relevant) is not at the fifth site but was associated with the octameric repeats.


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  		TABLE 2
Affinity of copper and manganese for PrP when metal binding sites are occupied

Shown are the log of the affinity values (M–1) for each site detected as determined from ITC experiments in which MnSO4 or CuSO4 (glycine chelate) was titrated into a solution of PrP protein at pH 5.5 buffered with 5 mM MES. Proteins used were either refolded in the presence of MnSO4 (manganese-saturated), CuSO4 (copper-saturated), or no metal (WT-apoPrP). ND, not detected. — indicates no experiment was performed.

 
We also assessed copper binding to manganese-saturated PrP with ITC. Studies of copper binding to PrP at four sites at pH 5.5 indicate four binding sites with a range of affinities as shown in Table 2. However, when saturated with manganese, only two sites could be detected with ITC. This implies that manganese bound to PrP blocked copper binding at two sites at pH 5.5. The H95A mutant showed the same number of sites as the wild-type protein, but the H110A and H95A/H110A mutants showed the loss of the high affinity copper binding site. This implies that in manganese-saturated PrP copper can bind at His-110 and somewhere in the octameric repeat region. Despite this result the PrP90–231 mutant showed no binding of copper or manganese when first loaded with manganese during refolding. This would suggest that metal binding at the fifth site is different in the absence of the amino acid residues 23–89 that include the octameric repeat region.

Manganese Binding to Copper-saturated PrP—It has been suggested previously that when manganese binds in place of copper it can alter the conformation of PrP. However, it has not been determined whether manganese can bind to PrP when it already binds copper. Manganese was titrated into PrP refolded in the presence of copper. The copper sites in PrP were filled with copper, but ITC experiments suggest that WT-PrP was still able to bind two atoms of manganese (Fig. 3 and Table 2). This result suggests that copper binding to PrP does not prevent manganese binding. In comparison the H95A mutant lacked the high affinity manganese site, whereas H110A did not. This implies that for copper-saturated PrP manganese is able to bind at the fifth site via His-95. Interestingly PrP90–231 saturated with copper showed no binding of manganese. This suggests that in the absence of the N terminus coordination of metal binding to the fifth site is altered.

We used CD spectroscopy to look at changes in the secondary structure of PrP on binding of manganese to copper-saturated PrP. Fig. 4 shows typical CD spectra from WT-PrP. Freshly prepared copper- and manganese-saturated WT-PrP produced similar CD spectra characterized by a deep minimum between 210 and 225 nm indicative of {alpha}-helical content. In contrast WT-PrP saturated with copper with subsequent manganese binding resulted in an altered spectrum with reduced helical content. This suggests that manganese binding to PrP results in an altered protein conformation.

Electrochemical Analysis of Manganese Bound to PrP—Voltammetric experiments were conducted in aqueous solution containing 5 mM MES buffer at pH 7. We have shown previously that PrP adsorbs onto edge plane pyrolytic graphite or boron-doped diamond electrode and results in immediately obvious and highly reproducible reversible current responses. Experiments could not be conducted at pH 5.5 as the copper centers would not remain strongly bound to the protein at this pH. Because of considerably lower capacitive background current using a boron-doped diamond electrode, this form of electrode was chosen for further examination. WT-PrP that had been saturated with either copper or manganese was examined as well as copper-refolded protein that had been exposed to manganese or manganese-refolded protein that had been exposed to copper. There was no obvious difference between the latter two conditions, so only one trace is shown. Fig. 5 shows the voltammograms obtained with a scan rate of 1 mV s–1. Fig. 5II shows the background as a dotted line, and a clear difference can be observed that can be attributed to reduction and oxidation by the manganese or copper centers. The midpoint potential for the copper- and manganese-saturated proteins was also determined. This is defined by Equation 1.

Formula 1(Eq. 1)
The redox potential for PrP binding copper was –0.01 V versus saturated calomel electrode, whereas the redox potential for PrP binding manganese was +0.08 V versus saturated calomel electrode.


Figure 2
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  		FIGURE 2.
Metal binding to manganese-saturated WT-PrP. Examples of ITC isotherms from different experiments are shown. A, MnSO4 was titrated into WT-PrP refolded in the presence of manganese. The resulting isotherm indicated one manganese binding site. B, CuSO4 was titrated into manganese-saturated WT-PrP. The isotherm indicated two copper binding sites. C, CuSO4 was titrated into manganese-saturated PrP-H95A. The isotherm indicated two copper binding sites. D, CuSO4 was titrated into manganese-saturated PrP-H110A. The isotherm indicated one low affinity copper site.

 
The peak current ratio on protein containing copper only was 1, suggesting a chemically fully reversible reaction for this condition. As the starting current and voltage was ~0, this reaction represents the oxidation of Cu+ to Cu2+ and its subsequent reduction back to Cu+. Where protein with only manganese was tested, the oxidation peak was much larger than the corresponding reduction, suggesting a partially irreversible oxidation of manganese. Subsequent cycles demonstrated an oxidation of equal intensity along with a lesser reduction equivalent to the initial cycle. This is likely to represent an oxidation of manganese from Mn2+ to Mn3+ and Mn4+. In addition, the effect of electron transfer kinetics is apparent by the wide separation between peaks. Protein that had been exposed to both manganese and copper showed a significantly smaller oxidation/reduction corresponding to the copper centers, suggesting that manganese had displaced some of the bound copper and thus reduced the copper redox signal.

The copper-bound protein contains five copper centers, four localized relatively closely together in the octarepeat unit and one within the fifth site. The oxidation signal produced for the wild-type copper-refolded protein corresponded to an integrated peak charge of 270 nanocoulombs. For the copper/manganese-refolded protein, this was reduced to 110 nanocoulombs for the copper signal and 90 nanocoulombs for the manganese signal. The manganese-only protein showed an integrated peak charge of 125 nanocoulombs. Experiments with pure mPrP protein in the absence of copper did not show this signal. As this charge is directly related to the amount of metal available for electron transfer on the protein, it would appear that three atoms of copper are displaced by manganese in the copper/manganese-binding protein.

Seeded Aggregation—One of the characteristics of PrPSc is the ability of the protein to catalyze aggregation of further PrP. In this regards "seeds" of PrP can be used to nucleate further aggregation. We have shown previously that recombinant PrP binding manganese can act as a seed to catalyze aggregation of metal-free recombinant PrP (26). As we have shown that manganese can bind to copper-saturated WT-PrP and alter its conformation we wished to determine whether manganese-binding PrP generated this way can also cause aggregation. Manganese-saturated and copper-saturated PrP were prepared and dialyzed free of unbound metals. The seeding material was added to freshly prepared metal-free PrP. As shown in Fig. 6, manganese-saturated PrP was able to stimulate aggregation as indicated by increased absorbance in a turbidity assay. Copper-saturated PrP was not able to stimulate aggregation. Copper-saturated PrP was then titrated with MnSO4 as for ITC experiments. Excess manganese was removed by dialysis. Manganese-binding PrP generated this way was able to stimulate PrP aggregation. This result suggests that binding of manganese can convert PrP to an aggregation-promoting protein even when copper is previously bound to the protein.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The prion protein is now well recognized as a copper-binding protein (34). It has also been suggested to bind manganese (8, 15). Previous studies have shown that PrP isolated from brains or from cultured cells has manganese bound (8, 35, 36). Until this report there has been no estimate of the affinity of free manganese for full-length PrP. Our results show that mouse PrP bound manganese at two sites. The two sites were detectible across a range of pHs. The optimum pH for manganese binding was pH 5.5. This suggests that manganese may bind to PrP in a more acid compartment of the cell such as endosomes. At this pH both sites would be occupied when the protein is exposed to micromolar concentrations of manganese. Unlike other metals such as copper, affinity values for manganese binding to known manganese-binding proteins are quite low and in the micromolar range (37–43). Divalent metal transporter-1 (DMT-1) is the only known transporter for Mn2+. The affinity of manganese for this protein has not been determined, but kinetic studies of manganese uptake suggest that its Km is 3 mM, suggesting a similarly low affinity (44). Therefore our values for the affinity of manganese and PrP are in the range expected for a manganese-binding protein. However, the concentration of manganese in mammalian brain is lower than this value (in the nanomolar range) (45). Based on this value it would be assumed that PrP would not bind manganese in vivo. If this were true then very few of the known manganese-binding proteins would bind manganese as they have similar affinities for the metal. Therefore, it is likely that manganese binding to proteins is regulated in a manner that is currently not understood. This metal has no known specific transport protein in the blood and has been suggested to enter cells through DMT-1, a protein known to transport a variety of divalent cations across the plasma membrane (46). Given the deficits in understanding of manganese metabolism, it is likely that the understanding of how manganese-binding proteins are able to associate with manganese will have to wait until more has been discovered of this system.

Previous assessment of manganese affinity for a recombinant PrP was for human PrP91–231. In this case a dissociation constant of 202 µM was determined (11). We also determined the affinity for a similar protein, mouse PrP90–231. However, the affinity we measured for this protein suggests a dissociation constant of 63 µM. This affinity was equivalent to the high affinity site we measured in the full-length protein. Our previous work suggested that full-length PrP bound four atoms of manganese per molecule (8). These new findings suggest that only two atoms bind per molecule. The difference between the two sets of data relates to the methods used to associate manganese with PrP. In the former experiments the metal was incorporated during refolding and appeared to associate with the octameric repeat region. In the latter findings metal was incorporated after PrP was refolded. The current study used a variety of mutants and peptides to determine the metal binding sites, and also the amount of metal bound was confirmed with cyclic voltammetry. Therefore we feel that these data are a truer reflection of metal binding as it would occur in vivo.

Studies with recombinant PrP have shown previously that binding of manganese results in a gradual change in protein conformation (8, 25). Initially PrP binding manganese shows the same conformation of PrP binding copper (see Fig. 4) but over time shows increased β-sheet content and proteinase K resistance (8). We have shown here that this change in structure can occur even when PrP still binds copper, implying that substitution of copper with manganese is not necessary. The change in structure of PrP on binding manganese has been verified recently using optical Raman spectroscopy (47). Other studies have shown protein conversion in mammalian cells and yeast cells when high levels of manganese are applied exogenously (8, 29). Protein conversion has also been observed in protein extracts from brains when manganese is added (27, 48).


Figure 3
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  		FIGURE 3.
Manganese binding to copper-saturated mutant PrP. Examples of ITC isotherms from different experiments are shown. A, MnSO4 was titrated into WT-PrP refolded in the presence of copper. The resulting isotherm indicated two manganese binding sites. B, MnSO4 was titrated into copper-saturated PrP90–231. No isotherm was produced, and no manganese binding sites were indicated. C, MnSO4 was titrated into copper-saturated PrP-H95A. One low affinity manganese binding site was indicated. D, MnSO4 was titrated into copper-saturated PrP-H110A. Two manganese binding sites were indicated.

 
Studies of manganese binding to peptides based on the sequence of PrP have produced variable results. In particular some studies suggest that manganese binds to peptides related to the octameric repeat region (11), and some suggest it does not (33, 49). Regardless of this, any measured affinity at this site was quite low. Our findings confirm this, as the octameric peptide showed binding of only one atom per molecule. This low affinity site was present in measurements with full-length protein. Manganese remained bound at the low affinity site despite extensive dialysis. Our cyclic voltammetry data also confirm that PrP binds two atoms but also show irreversible oxidation of the manganese centers on the protein. This would suggest a change in the valence state of the metal. In this case there could be a change in the dissociation constant for bound manganese explaining why it does not simply fall off the protein after extensive dialysis.

Research using peptides covering or overlapping the region 90–120 have been more useful in confirming manganese binding to PrP. 1H NMR studies with a peptide related to amino acid residues 106–126 of the human PrP sequences suggested coordination of manganese by Gly-126, Leu-125, Gly-124, and His-111 of this sequence (22). Our studies with the region 90–120 also showed manganese binding with an affinity similar to that reported for the 106–126 fragment (22). However, our studies suggested that mutating the histidine of this sequence (His-110 in mouse) had little effect on manganese binding and that His-95 played a much greater role in coordinating manganese binding. We also observed strong binding to peptide 90–120 but only at one site. The differences between these results cannot be attributed to pH as the experiments of Gaggelli et al. (22) were performed at pH 5.7, which is close to that used on our experiment (pH 5.5). Therefore, as the 106–126 peptide lacks His-95 it is possible that in the full-length protein manganese is inhibited from binding at His-110 due to structural constraints or electrostatic properties of the surround amino acid residues.

Our studies with peptide 90–120 showed an affinity higher than that of the full-length wild-type protein. One possible reason for this is that the greater potential flexibility of the shorter peptides allows the formation of stronger bonds or abrogates repulsive forces present in the full-length protein.

It is interesting to note that copper and manganese bind to different histidines within the fifth site in the full-length protein. Manganese binds at His-95, whereas copper binds at His-110. When studying the truncated mutant PrP90–231, this preference is probably lost as occupancy of the fifth site by one metal excludes binding of the other at this site. This implies a change in coordination of both metals within the fifth site in the truncated protein. In this alternative binding mode manganese could either bind to both histidines or associate with the protein as suggested by Gaggelli et al. (22).


Figure 4
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  		FIGURE 4.
CD analysis. Analysis of WT-PrP prepared with different metal content using CD is shown. Samples are taken from ITC experiments where metals were titrated into the protein. The samples were dialyzed before analysis. Spectra are shown for copper-saturated (thick line) and manganese-saturated (thin line) protein. In comparison WT-PrP refolded to bind copper and then exposed to manganese in the ITC experiment showed a different spectrum (medium line).

 


Figure 5
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  		FIGURE 5.
Cyclic voltammetry analysis. Shown is a cyclic voltammogram comparing wild-type PrP binding either copper (I), copper and manganese (II), or manganese (III). The protein was adsorbed onto a 3-mm boron-doped diamond electrode by incubation in 20 µM protein solution, pH 7, for 60 s. The scan rate used was 1 mV/s in an oxygen-excluded buffer of 5 mM MES, pH 7, at 25 °C. The dashed line on scan II represents the background signal where protein without metal is adsorbed to the electrode. E/V, electron/volts; SCE, saturated calomel electrode.

 


Figure 6
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  		FIGURE 6.
Seeded aggregation. Fresh, metal-free WT-PrP at 1 mg/ml was placed in a cuvette in a spectrophotometer. PrP seeds were added to the solution to a final concentration of 0.1 mg/ml. Change in absorbance was then assessed over 60 min at 325 nm. Seed samples were manganese-saturated PrP (squares), copper-saturated PrP (circles), or copper-saturated PrP exposed to manganese (triangles). An increase in absorbance is indicative of aggregation. The data shown are an example from experiments that were repeated four times.

 
The affinity of PrP for copper is much higher than for manganese (11). We previously suggested that manganese could occupy copper binding sites and compete equivalently for these sites (8). The current research suggests that manganese will occupy different sites than copper (as determined by ITC) but can cause the displacement of copper (from cyclic voltammetry data). This opens the possibility that binding of manganese to PrP can occur under any conditions and does not require the availability of copper-depleted protein. There is strong evidence that PrP can be isolated with manganese bound from both brain (36) and also from cells exposed to high non-toxic levels of manganese (8, 29). The consequence of such interactions is a change in conformation of the protein as observed here and reported previously (8, 25). Initially manganese binding does not result in an altered conformation, but over time, the protein is more susceptible to oxidative damage than metal-free or copper-binding protein that possibly results in changes in the structure of the protein (25). The conformation change in copper-charged PrP on exposure to manganese did not show this time dependence. This suggests a more specific effect that may include the copper bound to the protein as well. Interactions between PrP and copper under certain conditions have also been shown to cause changes in PrP such as increased proteinase K resistance (17).

The importance of manganese to research into prion disease touches many aspects of the field. Most importantly, manganese is bound to PrP isolated from the brains of patients with Creutzfeldt-Jakob disease (35). PrP with manganese bound is able to initiate seeded polymerization of metal-free prion protein (50). This ability is retained by the protein even when the metal is extracted from the protein. The current study has expanded on these findings by showing that PrP with manganese bound is able to seed PrP polymerization even if the protein already has copper bound. This suggests that in vivo binding of manganese to PrP does not require any alteration in its copper binding capacity. In the presence of hydrogen peroxide, binding of manganese or copper at the fifth metal binding site results in cleavage of the protein within the octameric repeat domain (26). PrP with manganese bound also has other qualities of the PrPSc. It becomes protease-resistant (8, 29), and it is neurotoxic (51).

In summary, we have identified the manganese binding sites in the prion protein and shown that the high affinity site is associated with His-95 in the so-called fifth metal binding site. We have shown that the affinity for this site is high for a manganese-binding protein and clearly implies that PrP could bind manganese in vivo. We also have shown that manganese bound to PrP becomes oxidized and is able to displace copper that may already be bound to the protein. As manganese binding to PrP is detrimental and causes a conformation change in the protein, its ability to bind to PrP when copper is still bound suggests that manganese binding could potentially play a role in prion disease progression in vivo.


   FOOTNOTES
 
* This work was supported by funding from the European Commission Quality of Life 5th Framework Programme (Grant QLRT-2001-02723) and the Biotechnology and Biological Sciences Research Council. 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. Back

1 To whom correspondence should be addressed: Dept. of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK. Tel.: 44-1225-383133; Fax: 44-1225-386779; E-mail: bssdrb{at}bath.ac.uk.

2 The abbreviations used are: PrPc, cellular prion protein; PrP, prion protein; PrPSc, scrapie prion protein; DEPC, diethyl pyrocarbonate; ITC, isothermal titration calorimetry; MES, 4-morpholineethanesulfonic acid; WT-PrP, wild-type mouse PrP. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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