Identification of the Cu²⁺ Binding Sites in the N-Terminal Domain of the Prion Protein by EPR and CD Spectroscopy

Abstract

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60–91. This region selectively binds divalent copper ions (Cu²⁺) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu²⁺–peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu²⁺ binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu²⁺/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu²⁺ is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu²⁺ is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.

Prion diseases are fatal, neurodegenerative disorders in which the pathogenic agent is an isoform of a monomeric, membrane-anchored glycoprotein called the prion protein (PrP^C).¹ Conversion of PrP^C to the multimeric, protease-resistant scrapie isoform (PrP^Sc) is the key pathogenic event (1–3). Fundamental questions remain concerning the physiological role of PrP^C, the mechanism leading to formation of PrP^Sc, and the neurotoxocity of this misfolded species.

The tertiary structures of recombinant unglycosylated human, mouse, and hamster PrP^C have been determined by NMR (4–8). Under the conditions used in these NMR studies, the N-terminal portion of mature PrP^C, residues 23–125, is largely unstructured in solution while the C-terminal segment, residues 126–231, shows considerable structure, including three α-helices and two small β-strands. Purification of PrP^C from brain homogenates reveals the full-length mature glycosylated and glycosylphosphatidylinositol-anchored (GPI) structure (residues 23–231) suggesting that residues 23–125 are likely to adopt a more ordered structure in vivo thereby avoiding additional proteolytic processing (9). The precise tertiary and quaternary structure of PrP^Sc is unknown, but spectroscopic studies show that it possesses high β-sheet content as well as residual helical structure (9, 10).

Recent evidence indicates that PrP^C may play a role in copper homeostasis (11–13). A number of studies have demonstrated that N-terminal fragments of PrP^C and the full-length protein bind divalent copper (Cu²⁺) (11, 14–19) and do so preferentially over other divalent metal ions (14, 15, 19). The N-terminal region of human PrP^C contains four sequential copies of a highly conserved octarepeat sequence PHGGGWGQ spanning residues 60–91 (20, 21). The high histidine content of this region suggests that it may be involved in metal ion binding (22). Spectroscopic studies have shown that Cu²⁺ induces ordered structure in the regions of PrP^C previously thought to be flexible by NMR spectroscopy (15).

Three models of the Cu²⁺ binding mode in PrP^C have been put forth based on spectroscopic studies. According to Stöckel et al. (15), who studied recombinant unglycosylated PrP(23–231) and various PrP fragments, at pH 6 two histidyl imidazole nitrogens and two glycine carbonyls from adjacent octarepeat units bind a single Cu²⁺. Viles et al. (17) observed cooperative binding in a variety of octarepeat containing PrP fragments. This led to the proposal that at just above pH 7, PrP(58–91) binds Cu²⁺ through a ring-like structure composed of four metal ions and four bridging imidazolate ions. Alternatively, Raman studies by Miura et al. (16, 18) on single and multi-octarepeat containing peptides suggest that each octarepeat independently binds Cu²⁺ and that the minimum binding unit is the HGGG segment of the octarepeat. Here, the N_π nitrogen (also denoted Nδ1) of the imidazole and the amide nitrogens of the last two glycine residues coordinate Cu²⁺.

While each study proposes a different structural model, there is good agreement regarding the Cu²⁺ to octarepeat binding stoichiometry. All work to date on N-terminal segments above pH 7 show that the stoichiometry is 1:1 Cu²⁺/octarepeat (11, 14, 16–18) with the exception that Viles et al. find that PrP(51–75) and PrP(73–91), each of which contains two octarepeat units, bind a single copper ion (17). Stöckel et al. find that, below pH 7, in full PrP two octarepeats are necessary to bind a single Cu²⁺ ion.

Given that the octarepeat repeat is highly conserved among mammalian PrP proteins, and further that PrP binds Cu²⁺ in vivo (11), strongly suggests that this region plays a physiological or structural role. Efforts to determine the precise function of PrP are ongoing (13, 23–25). Brown et al. (23) have shown that PrP^C folded in the presence of Cu²⁺ exhibits superoxide dismutase (SOD) activity and may serve to protect synaptic regions from oxidative stress. However, PrP^C is continuously recycled through endocytosis and, thus, an alternative proposal is that the role of PrP^C is to shuttle Cu²⁺ from the synaptic space to the cell interior (13, 19, 25). Indeed, recent work suggests that Cu²⁺ stimulates endocytosis of PrP (13) and, further, that PrP may release Cu²⁺ in the low pH environment of endosomes (19). Characterization of the Cu²⁺ binding site and evaluation of whether such a binding site will be pH-sensitive in vivo are essential for clarifying PrP’s physiological function.

Divalent copper is a d⁹ metal ion and is readily observable by electron paramagnetic resonance. We report an EPR study detailing the magnetic properties of copper-bound peptide fragments corresponding to one, two, and four PrP octarepeats and a series of six peptides that are shortened or frame-shifted (with respect to the octarepeat peptide sequence) analogues of the conserved octarepeat unit. In addition, a peptide of four octarepeats where the tryptophan side chains were replaced with cyclohexylalanine, a bulky nonaromatic residue, was examined. pH titrations followed by EPR show that Cu²⁺ is bound at pH values greater than 5. At pH 7.4, the 1, 2, and 4 peptide repeats have nearly identical EPR spectra indicating that the mode of binding is similar in all three. Two EPR active binding modes were observed. The dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion. This is supported by S-band EPR which directly reveals ¹⁴N superhyperfine couplings. ESEEM spectra demonstrate that one of the nitrogens is from the histidine imidazole. EPR spectra of Cu²⁺ bound HGGGW, corresponding to only five residues of the octarepeat, suggests that this peptide is the minimum unit capable of mimicking the dominant coordination environment of the octarepeat and longer N-terminal peptides. However, EPR of Cu²⁺ bound to peptide HGG reveals the existence of both binding modes, and this sequence may therefore contain the nitrogens that directly bind copper. CD spectra obtained in the visible region corresponding to the d–d transitions revealed only small variations of the associated λ_max values. The stoichiometry of Cu²⁺ binding was determined using both EPR and CD spectroscopy and, consistent with previous studies, demonstrates that the Cu²⁺/octarepeat stoichiometry is 1:1. Finally, EPR spin quantitation of the signal arising from Cu²⁺ bound to a four octarepeat peptide suggests that all of the bound copper ion is detected. This finding is of relevance when considering proposed structures containing bridging imidazolates since such bridging results in a reduction of the EPR signal.

MATERIALS AND METHODS

Peptide Synthesis and Purification

PrP(57–91), PrP(23–28, 57–91), PrP(23–28, 57–91 W → X), and PrP(73–91) were synthesized at UCSF. All other peptides were prepared at UCSC by solid-phase synthesis using standard fluorenylmethoxycarbonyl (Fmoc) methods. Peptides were acetylated at the N-terminus and amidated at the C-terminus. Finally, all peptides were purified using reverse phase HPLC and characterized by electrospray ionization mass spectrometry (ESI-MS) (VG Quattro II, Fisons Instruments).

Peptide Sample Preparation and Concentration Determination

All peptide solutions were made in a degassed buffer containing 25 mM N-ethylmorpholine (NEM), 150 mM potassium chloride (KCl), and 20% glycerol (v/v) where the glycerol served as a cryoprotectant. The pH of the solution was adjusted to the desired value using a concentrated hydrochloric acid (HCl) or potassium hydroxide (KOH) solution. This buffer was used for all studies except where otherwise indicated. Peptide concentrations were determined by acquiring UV–Vis spectra of samples in 6 M guanidine hydrochloride. The extinction coefficient for tryptophan at 280 nm, which was taken to be 5609 M⁻¹ cm⁻¹, was used to calculate the peptide concentration.

All samples for EPR experiments, with the exception of those used for Cu²⁺ titrations, were prepared with ⁶³CuCl₂ (99.62%, Cambridge Isotope Laboratories). The pure isotope ⁶³Cu was used to avoid inhomogeneous broadening of the EPR lines that would otherwise be present with the mixture of naturally occurring isotopes ⁶³Cu and ⁶⁵Cu.

Electron Paramagnetic Resonance (EPR) and Electron Spin–Echo Envelope Modulation Spectroscopy (ESEEM)

X-Band EPR spectra (9.43 GHz) were acquired using a Bruker ESP380 spectrometer and a TE₁₀₂ cavity equipped with variable temperature control. Unless otherwise specified, all X-band EPR spectra were acquired at 77 K. Spectra at liquid helium temperatures were acquired using a dielectric resonator (9.73 GHz). S-band spectra (3.5 GHz) were acquired at 133 K using a loop gap resonator as part of a specially designed spectrometer housed at the Biomedical ESR Center at the Medical College of Wisconsin. The m_I = −1/2 lines of these spectra were simulated using stick diagrams with Gaussian line shapes. Three pulse ESEEM measurements were obtained at 4.2 K on an X-band pulsed-EPR spectrometer. The instrument, cavity, and resonator were constructed in-house and have been previously described (26, 27). Continuous wave spectra were first acquired on a Varian E-112 X-band spectrometer. Data were then obtained at g_⊥, the point of greatest spectral intensity (3315 G at 9.47 GHz). The ESEEM acquisition parameters were τ = 150 ns, starting T = 40 ns, a time increment of 20 ns, and a repetition rate of 53 Hz. Data processing to attain frequency domain spectra was carried out using software described in previous work (28).

Circular Dichroism (CD) Spectroscopy

CD spectra were acquired on an Aviv 60DS spectrometer at 298 K. A 1-cm path length cell was used for spectra recorded between 350 and 800 nm sampling points every 1 nm.

Titration with Cu²⁺

Copper titrations were monitored by EPR at 77 and 298 K and by CD at 298 K. Solutions of Cu²⁺ were made from CuCl₂ such that 5-µL additions would deliver the equivalents of copper necessary for the given titration. The pH of the copper stock was adjusted to 7.4 so that the pH of the peptide solution would not drop during the titration due to the addition of the Cu²⁺ solution. Copper concentrations were determined by comparison to standard solutions containing known amounts of Cu²⁺ in 10 mM imidazole at pH 7.4. Foldover concentrations, which were used to determine the binding equivalence, were determined by fitting the low and high [Cu²⁺] regions of the curve to a linear function and then evaluating the point of intersection.

RESULTS

EPR and CD

The peptide segment PrP(57–91) contains four repeats of the conserved sequence PHGGGWGQ and corresponds to the main N-terminal copper binding region as suggested by other studies (14, 15). Our strategy focused on examining a series of peptide segments derived from PrP-(57–91) to determine the structural elements necessary for Cu²⁺ binding. Table 1 displays all peptides investigated. Several of the sequences contain the string of basic residues KKRPKP at their N-termini corresponding to PrP(23–28). As will be discussed further below, addition of this sequence increased solubility in aqueous solution which, in turn, facilitated spectroscopic measurements. All of the peptides are composed of only standard l-amino acids with the exception of PrP(23–28, 57–91 W → X) where all tryptophan side chains were substituted with the nonaromatic cyclohexylalanine moiety. This peptide was included to explore the role of the indole side chain in Cu²⁺ coordination. All peptides listed below the octarepeat in Table 1 are truncated and/or frame-shifted analogues of this basic unit. Finally, all peptides were chemically blocked with an acetyl group (Ac) at the N-terminus and an -NH₂ group at the C-terminus. Blocking the N-terminus was important since polypeptide N-terminal amines can bind directly to copper ions.

Table 1.

PrP-Derived Peptide Sequences

PrP(57–91)	WGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQ
PrP(23–28, 57–91)	KKRPKPWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQ
PrP(23–28, 57–91 W→X)	KKRPKPXGQPHGGGXGQPHGGGXGQPHGGGXGQPHGGGXGQ
PrP(73–91)	PHGGGWGQPHGGGWGQ
PrP(23–28, 73–91)	KKRPKPWGQPHGGGWGQPHGGGWGQ
Octarepeat	PHGGGWGQ
	GQPHGGGW
	GQPHGGG
	HGGGWG
	HGGGW
	HGGG
	HGG

It is well-established that Cu²⁺/peptide interactions are strongly pH-dependent. The X-band EPR spectra of PrP-(23–28, 57–91) with 4 equiv of ⁶³Cu are shown in Figure 1 at several pH values between 4 and 12. Figure 1 shows that as the pH is increased there are marked changes of the four hyperfine lines in the low field, parallel region of the spectrum. (Note, the four lines in this region arise from hyperfine coupling to the I = 3/2 ⁶³Cu nucleus.) Specifically, g_‖ and A_‖ shift, and this is readily ascribable to changes in the ligand environment around the copper ion (29). Figure 2 shows an expansion of this low field region at selected pH values. At pH 4.10, the spectrum has four dominant lines with g_‖ = 2.41 and A_‖ = 408 MHz (121 G), which is characteristic of aqueous copper. This aquo signal suggests there is no detectable binding between Cu²⁺ and peptide at this pH. At pH 5.30, a new set of lines appear corresponding to a spectrum with g_‖ = 2.37 and A_‖ = 415 MHz (125 G). According to established Peisach–Blumberg correlations, these values clearly fall in the range expected for coordination of a single nitrogen ligand and three oxygen ligands in the equatorial plane (29). The observation that binding begins to occur near pH 6 supports previous suggestions that histidine is involved in complexing Cu²⁺ (15–18). Increasing the pH to 6.75, the spectrum is now composed of two sets of lines with values g_‖ = 2.27, A_‖ = 537 MHz (169 G) and g_‖ = 2.23, A_‖ = 493 MHz (158 G), respectively. At pH 8.57, only the latter spectrum is observed. Both of these spectra are consistent with binding of 2–3 nitrogen ligands. Finally, at pH 11.60, only one species is observed with g_‖ = 2.20 and A_‖ = 582 MHz (189 G), which is consistent with binding to four nitrogen ligands. Magnetic parameters derived from these spectra are summarized in Table 2.

Figure 1.

X-band EPR spectra of ⁶³Cu²⁺ loaded PrP(23–28, 57–91) at different pH values. The grid at top identifies the four hyperfine lines arising from coupling to the ⁶³Cu (I = 3/2) nucleus. All spectra were collected at 77 K, ν_o = 9.43 GHz, and a sweep width of 1200 G.

Figure 2.

Expansion of the low field (parallel) region from Figure 1 at selected pH values. As the pH increases there are marked changes of the four hyperfine lines reflecting changes in the ligand environment of the copper ion. Distinct species apparent at various points in the pH range are indicated.

Table 2.

EPR Parameters for ⁶³Cu²⁺ Loaded PrP(23–28, 57–91) at Different pH Values (cf. Figures 1 and 2)

		A‖b
pH	g‖a	MHz	(G)	g⊥a	ligating atoms
4.10	2.41	408	(121)	2.09	4 O
5.30	2.37	415	(125)	2.08	3 O, 1 N
6.75	2.27	537	(169)	2.06	2 O, 2 N
7.45	2.23	493	(158)	2.06	1 O, 3 N
8.57	2.23	493	(158)	2.06	1 O, 3 N
11.60	2.20	582	(189)	2.05	4 N

^aThe error in all reported g-values is less than ± 0.01.

^bError in A values ± 6 MHz (± 2 G).

Returning to Figures 1 and 2, the spectrum obtained at pH 7.45 shows that the dominant copper bound PrP(23–28, 57–91) species is the same as that observed at pH 8.57. We refer to this spectrum as component 1 (Figure 2). Also, the lines observed at pH 6.75 are still present as a minor spectral component at pH 7.45, demonstrating that at least one other binding mode is present. We refer to this second spectrum as component 2. As noted above, component 1 and component 2 spectra are not absolutely definitive with respect to the number of bound nitrogens based on magnetic parameters alone. However, given that nitrogen ligation in peptide–Cu²⁺ complexes increases with elevated pH, we assign component 1 as arising from coordination with three nitrogens and one oxygen and component 2 arising from coordination with two nitrogens and two oxygens.

Figure 3 displays the EPR spectra of all the peptides with excess ⁶³Cu²⁺ at pH 7.45 ± 0.07. Excess copper was used to ensure that all peptides were fully loaded with metal ion. At pH ≥ 7, aquo Cu²⁺ is only sparingly soluble and precipitates as EPR silent [Cu(OH)₂]_n, and hence the excess of Cu²⁺ does not introduce interference from the aquo species. The spectrum of PrP(57–91) is shown at the top of Figure 3. This peptide was less soluble than PrP(23–28, 57–91) when bound as a copper complex and signal averaging was required to obtain a high quality spectrum. (Note: this sample contained less than 100 µM peptide.) The splitting between the m_I = −1/2 and m_I = +1/2 hyperfine lines serves as a diagnostic for assessing g_‖ and A_‖, and thus two vertical lines passing through these features of component 1 have been added to Figure 3 to aid in comparison of the spectra. The spectrum obtained from PrP(57–91) is nearly identical to that of PrP(23–28, 57–91) with all the transitions appearing in the same positions with similar intensity ratios. The Cu²⁺/peptide system PrP(23–28, 57–91 W → X) also produces a spectrum where the main hyperfine lines are nearly superimposable with Prp(23–28, 57–91). The similarity observed between PrP(23–28, 57–91) and PrP(23–28, 57–91 W → X) suggests that the indole side chain has minimal influence on the environment of the paramagnetic electron.

Figure 3.

X-band EPR spectra of all ⁶³Cu²⁺/peptide complexes at pH values 7.45 ± 0.07. Spectral component 1 and component 2 are indicated with grids at top. Vertical lines have been added to the m_I = −1/2 and m_I = +1/2 hyperfine lines of component 1 to aid in comparison of g_‖ and A_‖ values between complexes. All spectra collected at 77 K, ν_o = 9.43 GHz, and a sweep width of 1200 G.

The peptides PrP(73–91) and PrP(23–28, 73–91), containing two octarepeats, give spectra nearly identical to that of PrP(57–91). One interesting feature unique to these two sequences and PrP(23–28, 57–91) is a new component in the high field region of the spectrum at approximately 3500 G. The origin of this feature is not clear but could arise from dipolar coupling between closely spaced copper ions (30).

The octarepeat unit itself, PHGGGWGQ, and the frame-shifted analogue GQPHGGGW reproduce the spectral features observed for both PrP(57–91) and PrP(23–28, 57–91). The next system, GQPHGGG, which does not contain the tryptophan residue, reveals a change in the spectrum. As compared to PrP(57–91) and PrP(23–28, 57–91), component 2 is more pronounced. Comparing this spectrum to that obtained from the octarepeat suggests that a residue following the HGGG unit helps structure the coordination environment.

The peptides HGGGWG and HGGGW represent substantial reduction of the fundamental octarepeat sequence. Interestingly, spectra obtained from these peptides reproduce the spectral features of PrP(57–91). However, further reduction to HGGG yields a pronounced difference when compared to the octarepeat. Specifically, now component 2 dominates the spectrum. Finally, the tripeptide HGG exhibits approximately the same spectrum as HGGG. (When preparing Cu²⁺ complexes from these small peptides sufficient Cu²⁺ was added to ensure that the copper ion was bound by only a single peptide molecule, and this was verified with quantitative spin integration for HGGGW and all longer peptides. Thus, formation of dimeric species where two peptide molecules bind a single Cu²⁺ was avoided.)

Magnetic parameters obtained from the spectra in Figure 3 are reported in Table 3. Parameters for component 1 reveal little variation, although A_‖ is slightly reduced for those peptides without a residue following the HGGG segment. Parameters for component 2 spectra could only be reliably obtained for three of the peptides, but again variation among this set is small. Given that HGGGW is the shortest peptide to give only a component 1 spectrum suggests that this segment binds Cu²⁺ in a fashion equivalent to the longer multi-octarepeat peptides and therefore comprises the fundamental binding unit.

Table 3.

EPR Parameters for All ⁶³Cu²⁺/Peptide Complexes at pH values 7.45 ± 0.07 (cf. Figure 3)

	component 1			component 2
sequence @ pH values 7.45 ± 0.07		A‖b			A‖b
	g‖a	MHz	(G)	g‖a	MHz	(G)
PrP(57–91)	2.23	490	(157)
PrP(23–28, 57–91)	2.23	493	(158)
PrP(23–28, 57–91 W → X)	2.23	490	(157)
PrP(73–91)	2.23	495	(159)
PrP(23–28, 73–91)	2.23	493	(158)
PHGGGWGQ	2.23	499	(160)
GQPHGGGW	2.23	493	(158)
GQPHGGG	2.23	472	(151)	2.26	535	(169)
HGGGWG	2.23	492	(157)
HGGGW	2.23	496	(159)
HGGG	2.23	461	(148)	2.26	525	(166)
HGG	2.23	473	(152)	2.26	524	(166)

^aThe error in all reported g-values is less than ± 0.01.

^bError in A values ± 6 MHz (± 2 G).

PrP(57–91) was further analyzed to test for the presence of any coupled copper species. The half-field region of the spectrum was scanned for the ΔM = 2 transition that is often observed in the spectra of spin coupled complexes. No ΔM = 2 transition was found. EPR spectra were also collected at several temperatures down to 4 K, and again there was no evidence of a ΔM = 2 transition. Finally, we note that all copper could be accounted for by spin quantitation, and this will be discussed further below.

When Cu²⁺ is added to the peptides, CD bands arising from weakly allowed d–d transitions appear in the visible region. These transitions become observable by CD only when Cu²⁺ is in a chiral ligand environment, and thus trace amounts of aqueous copper ion are not detected. The CD spectra for the peptides at pH 7.4 are shown in Figure 4. For the systems PrP(23–28, 57–91), PrP(73–91), and PrP-(23–28, 73–91), these transitions are centered roughly at 650 nm with a positive band near 580 nm and a negative band near 700 nm. [Poor solubility of the copper complex of PrP(57–91) prohibited the acquisition of a CD spectrum with an acceptable signal-to-noise ratio, and thus it is not included in Figure 4.] The single octarepeat and all systems equal in size or smaller exhibit transitions centered at approximately 665 nm with positive bands near 600 nm and negative bands near 720 nm. The peptide PrP(23–28, 57–91 W → X) has CD characteristics that fall between those of the large and small systems with the transition centered at approximately 662 nm and positive and negative bands at 595 and 724 nm, respectively. An additional positive band is apparent at 340 nm for all copper/peptide complexes studied here (not shown).

Figure 4.

Visible region CD spectra of all Cu²⁺/peptide complexes at pH 7.4. The spectra are normalized along the y axis for comparison of λ_max and λ_min. The approximate baseline for each spectrum is indicated by hatch marks at the left. The λ_max of the positive CD band is listed to the right of each spectrum. Because of poor solubility, PrP(57–91) is not included in this figure. All spectra collected at 298 K.

Absorption bands due to d–d transitions can arise over a range of wavelengths from 500 to 750 nm. Transition pairs arising specifically between 570 and 750 nm are consistent with tetragonally distorted five- or six-coordinate Cu²⁺ with nitrogen ligands. It is interesting to note that while the EPR shows a clear difference between HGGGW, the proposed fundamental binding unit, and the shorter sequences HGGG and HGG, the optical transitions are indistinguishable. It appears that these short peptides all bind mainly through nitrogen coordination, but there are subtle differences in the local coordination geometry that is reflected mainly in the EPR spectra. The subtle shift of approximately 20 nm observed in the transitions when comparing the short peptides to the longer multi-octarepeat sequences is very small but may reflect somewhat greater tetragonal distortion for the longer sequences.

Cu²⁺ Titrations Followed by EPR and CD

EPR and CD signals were obtained as a function of titrated Cu²⁺ to determine metal ion to octarepeat binding stoichiometry. Our interest was in determining whether the stoichiometry differs between the octarepeat and the multi-octarepeat sequences. We therefore focused solely on the single octarepeat, PrP-(23–28, 73–91) with two octarepeats, and PrP(23–28, 57–91) with four octarepeats.

The positive CD signal intensity was followed as a function of added Cu²⁺ at pH 7.4. The titration curves for the octarepeat, PrP(23–28, 73–91), and PrP(23–28, 57–91) are shown in Figure 5. Peptide concentrations were between 50 and 400 µM and copper ion concentrations reported in equivalents per peptide (see Figure 5 for exact concentration of each peptide). Figure 5 shows that the CD band increases and folds over at 1.1 ± 0.2, 2.5 ± 0.5, and 4.4 ± 0.8 equiv of added copper for the three peptides, respectively. Interestingly, the titration curve for the octamer increases linearly, whereas the curves for both PrP(23–28, 73–91) and PrP(23–28, 57–91) show curvature in the region of 1.0 added equivalent. This is discussed further below. The maximum positive molar ellipticity was 2180 deg cm² dmol⁻¹ for the octarepeat unit, 4370 deg cm² dmol⁻¹ for PrP(23–28, 73–91), and 7572 deg cm² dmol⁻¹ for PrP-(23–28, 57–91). Normalizing such that the maximum positive molar ellipticity reached for the octarepeat unit is defined as 1.0 equiv of Cu²⁺, PrP(23–28, 73–91), and PrP-(23–28, 57–91) are found to bind approximately 2.0 and 4.2 equiv of Cu²⁺, respectively. Thus, CD signal titration and maximal signal intensity both demonstrate that the Cu²⁺/octarepeat stoichiometry is 1:1 regardless of the number of octarepeats in a given peptide. These results are summarized in Table 4.

Figure 5.

CD signal intensity at λ_max as a function of titrated Cu²⁺ at pH 7.4 for the octarepeat, PrP(23–28, 73–91), and PrP(23–28, 57–91). Peptide concentrations are 398, 333, and 51 µM, respectively, and copper ion concentrations are reported in equivalents per peptide.

Table 4.

Summary of EPR and CD Titration Data for the Octarepeat, PrP(23–28, 73–91), and PrP(23–28, 57–91) (cf. Figures 5 and 6)

	octarepeat (equiv)	PrP(23–28, 73–91) (equiv)	Prp(23–28, 57–91) (equiv)
max EPR	0.92	1.90	4.24
signal intensitya
Cu2+ conc	0.83	1.88	3.83
detected by EPRb
max CD	1.06	2.53	4.37
molar ellipticityc
normalized CD	1.00	2.00	4.19
molar ellipticityd

^aEquivalents of Cu²⁺ added to achieve maximum EPR signal intensity.

^bAbsolute bound Cu²⁺ concentration detected by EPR.

^cEquivalents of Cu²⁺ added to achieve maximum CD molar ellipticity.

^dMaximum molar ellipticity scaled as integer value of octarepeat molar ellipticity when fully loaded with Cu²⁺.

Double integration of first derivative EPR spectra was used to quantitate the amount of titrated Cu²⁺ at pH 7.4. Peptide concentrations were in the range of 250 to 1000 µM, and again the copper ion concentration is reported in equivalents per peptide. Results for the octarepeat, PrP(23–28, 73–91), and PrP(23–28, 57–91) are shown in Figure 6. The titration curves for these three systems all increase linearly and fold over at 0.9 ± 0.2, 1.9 ± 0.4, and 4.2 ± 0.8 equiv of added copper, respectively. The absolute concentration of bound Cu²⁺ was determined by spin integration followed by comparison to a copper standard. Quantitated in this manner, octamer, PrP(23–28, 73–91), and PrP(23–28, 57–91) bind 0.8 ± 0.1, 1.9 ± 0.3, and 3.8 ± 0.6 equiv of copper, respectively. These data agree with the CD results and again demonstrate a 1:1 Cu²⁺/octarepeat stoichiometry (see Table 4).

Figure 6.

Integrated EPR signal intensity as a function of titrated Cu²⁺ at pH 7.4 for the octarepeat, PrP(23–28, 73–91), and PrP-(23–28, 57–91). Peptide concentrations are 1000, 500, and 250 µM, respectively, and the copper ion concentration is reported in equivalents per peptide. All spectra collected at 298 K.

Returning to the CD titration in Figure 5, it was noted that the titration curves for both PrP(23–28, 73–91) and PrP(23–28, 57–91) are curved at low Cu²⁺ concentration, whereas for the octarepeat it is linear. Specifically, with 1 equiv of Cu²⁺, PrP(23–28, 57–91) gives a very weak CD signal relative to the octamer. In contrast, the EPR signal integrals in Figure 6 show that all added Cu²⁺ is bound in this same respective region of the titration curve. Affinities have been determined for PrP–Cu²⁺ complexes, and the dissociation constants (K_d) range from 1 to 5 µM. Accordingly, at the concentrations used for the CD and EPR titrations (>150 µM in octarepeat concentration), greater than 85% of the Cu²⁺ should be bound in a 1:1 complex, and this is consistent with the EPR titration data and the CD titration of the octamer. Thus, the weak CD signal at 1.0 equiv of Cu²⁺ observed for the longer complexes arises not from lack of association to form the peptide–Cu²⁺ complex but instead a molecular environment that exhibits lower CD activity than that of the octamer–Cu²⁺ complex. That this curvature exists only for the multi-octarepeat sequences argues that at low Cu²⁺ concentration there is a coordination environment that differs from that observed in the fully bound complexes. To test for this possibility, we examined EPR spectra of PrP(23–28, 57–91) at various Cu²⁺ concentrations, and the results are shown in Figure 7. Spectra are normalized such that the integrals are equal. Indeed, at 1 and 2 equiv, the spectra are markedly different than that observed at 4 equiv. The spectral features of the dominant species at 1 equiv (arrows) are similar to the component 2 spectra of Figure 3, perhaps suggesting that two nitrogens coordinate at low Cu²⁺ concentrations. Viles et al. have suggested that two His side chains may bind to the metal ion centers at low Cu²⁺ concentrations, and this may explain the results of Figure 7 (17) and the cooperative nature of Cu²⁺ binding (11, 19). How the coordination environment changes as a function of Cu²⁺ concentration is an issue of considerable importance given biological concentrations of Cu²⁺ (11), PrP (31), and other copper binding species.

Figure 7.

X-band EPR spectra of 133 µM PrP(23–28, 57–91) with 1, 2, 3, and 4 equiv of Cu²⁺ at pH 7.4. Each spectrum has been normalized to unit area. Arrows have been added above the m_I = −3/2 and m_I = −1/2 hyperfine lines to highlight changes in this region. All spectra collected at 77 K, ν_o = 9.43 GHz, and a sweep width of 1200 G.

ESEEM and S-Band EPR

Three pulse electron spin–echo envelope modulation (ESEEM) spectroscopy (32, 33) was performed to aid in the characterization of the Cu²⁺ coordination environment. Spectra for fully Cu²⁺ loaded PrP-(23–28, 57–91), octarepeat, and HGGGW are shown in Figure 8. PrP(23–28, 57–91) is less soluble than the other two peptides thereby accounting for the relative reduction in the signal-to-noise of its ESEEM spectrum. All three spectra are quite similar and are dominated by features that are consistent with coordination by the histidine imidazole. Specifically, the low-frequency lines at 0.59, 0.87, and 1.47 MHz are characteristic of a weakly coupled ¹⁴N close to the exact cancellation limit (33, 34). This limit corresponds to a zero vector sum of the ¹⁴N nuclear Zeeman and hyperfine fields in one of the electron spin manifolds and thus gives rise to nearly pure ¹⁴N quadrupolar transitions. These frequencies yield the quadrupolar parameters |e²Qq/h| = 1.56 MHz and η = 0.75 (26). The values of these parameters and the observed ESEEM frequencies are typical of that observed for the remote, noncoordinated nitrogen of an imidazole ring bound to Cu²⁺ (26). In addition, the broad features at approximately 4 MHz arise from the Δm_I = 2 transitions taking place within the other electron spin manifold (26, 33).

Figure 8.

Three pulse ESEEM spectra for PrP(23–28, 57–91), the octarepeat (PHGGGWGQ) and HGGGW obtained at X-band. Spectra were obtained at 4.2 K from the g_⊥ region of the spectrum with τ = 150 ns.

The ESEEM spectra in Figure 8 are typical for Cu²⁺ coordinated by imidazole and thus support the assignment here and in other works (14, 15, 17, 18) that the histidine side chain participates in the coordination environment. ESEEM spectra can also be used to assess the number of histidines bound to the copper center. Specifically, when two or more imidazoles coordinate to a single Cu²⁺, the Δm_I = 2 signal is enhanced and accompanied by combination peaks arising at multiples of the fundamental low frequency (<2 MHz) transitions (35). The relative amplitude of the Δm_I = 2 transition for PrP(23–28, 57–91) does not appear to be enhanced when compared to the same transition for the octarepeat and HGGGW. Furthermore, ESEEM of PrP(23–28, 57–91) does not reveal evidence of combination peaks between 2 and 3 MHz. Hence, these ESEEM spectra suggest that each octarepeat within PrP(23–28, 57–91) contributes a single imidazole to its coordinated Cu²⁺.

The values of the nuclear quadrupolar transitions also identify the protonation state of the remote nitrogen. The remote nitrogen of neutral imidazole gives rise to Cu²⁺ ESEEM spectra with sharp transitions below 2 MHz as observed here. Deprotonated imidazole (e.g., imidazolate) gives transitions at approximately 0.24 MHz, 2.34 MHz, and 2.58 (|e²qQ/h| ≈ 3.28 MHz, η ≈ 0.15) (33) and thus cannot account for dominant ESEEM transitions for the three peptides in Figure 8. Finally, transitions at 2.00 and 2.85 MHz are observed for the octarepeat and HGGGW. At this juncture, the origin of these transitions is not clear; however, they do not appear to arise from combinations of the lower frequency components. In addition, Cu²⁺ titration experiments with the octarepeat (Figures 5 and 6) demonstrate a 1:1 Cu²⁺ to peptide stoichiometry thereby ruling out multiple histidine coordination in these short peptides. It is possible that these peaks arise from other noncoordinated nitrogens within the HGGGW binding unit. Interestingly, these transitions are not clearly observed for PrP(23–28, 57–91). Perhaps subtle variations in the octarepeat structures within this four octarepeat peptide results in variations of these frequencies thereby broadening and reducing the relative amplitude of these lines. Aside from these two unassigned components, these ESEEM data suggest that the imidazoles in all three peptides exist in similar chemical environments and clearly participate in Cu²⁺ coordination.

Analysis of the component 1 EPR spectra in Figures 1–3 suggests that three nitrogens participate in the equatorial coordination environment of the Cu²⁺ center in all peptides that contain the fundamental HGGGW segment. The next goal is to test this assignment by probing for the existence of nitrogen superhyperfine couplings. Nitrogens directly coordinated to Cu²⁺ typically give couplings between 10 and 15 G (36, 37). Unfortunately, these couplings are often masked by inhomogeneous broadening arising from g and A strain. Spectra for the octarepeat and GQPHGGGW in Figure 3 do exhibit subtle superhyperfine structure near the first derivative maximum at 3250 G. However, analysis of couplings in this g_⊥ region of the spectrum is difficult because small differences in g_xx and g_yy can complicate the multiplet structure. Another approach is to employ low frequency S-band (3.5 GHz) EPR. The benefit of S-band EPR arises from a partial cancellation of g-strain and A-strain induced inhomogeneous broadening specifically for the ⁶³Cu m_I = −1/2 hyperfine line (36). The S-band EPR spectrum for the octarepeat is shown in Figure 9. Because of the reduced Zeeman interaction, the ⁶³Cu hyperfine interaction now dominates the spectrum. The m_I = −1/2 hyperfine line reveals subtle splittings arising from superhyperfine structure as seen in the full scan (trace A). To clearly resolve the couplings, the m_I = −1/2 hyperfine line was selectively scanned at high resolution with signal averaging (trace B). This signal was then differentiated to give the second harmonic (trace C) which emphasizes the multiplet splitting (38). Qualitatively, the splittings are approximately 13 G, which is consistent with ¹⁴N coupling from directly coordinated nitrogens. Indeed, these observed splittings are similar to that obtained from other Cu²⁺ centers with several coordinated nitrogens.

Figure 9.

S-band EPR (3.5 GHz) of the octarepeat (PHGGG-WGQ). The grid at the top shows the ⁶³Cu hyperfine splitting, and the m_I = −1/2 line is indicated. Trace A is the full field scan; trace B is the m_I = −1/2 line scanned with high resolution and signal averaging; trace C is the derivative of B emphasizing the multiplet structure.

S-band EPR spectra of the ⁶³Cu m_I = −1/2 line are shown for the octarepeat and HGGGW in Figure 10. To within experimental error, the observed multiplet patterns are equivalent. Each shows a splitting pattern consisting of six or more resolved lines. Interestingly, the symmetric splitting observed at the top of the first derivative peak demonstrates that the superhyperfine multiplets arise from an even number of lines. If the multiplets were strictly from equivalent ¹⁴N superhyperfine interactions, one would expect an odd multiplet pattern. For example, one ¹⁴N would give three lines (1:1:1 relative intensity), and two equivalent ¹⁴N’s would give five lines (1:2:3:2:1 relative intensity), etc. (39). The even multiplet observed here can arise from either inequivalent ¹⁴N’s or an additional I = 1/2 spin arising from perhaps a nearby ¹H (39). In most cases, directly coordinated nitrogens do not exhibit grossly different hyperfine couplings, and, alternatively, there is good precedent for participation of several nitrogens and a proton contributing to these multiplets (40). (Note that these spectra were obtained in D₂O, so an ¹H must arise from a peptide hydrogen.) Simulations were performed assuming equivalent ¹⁴N couplings (a_N) and coupling from a single ¹H (a_H). Limiting the simulations to reasonable values of these couplings, the best results were obtained from three nitrogens with a_N = 13 and a_H = 10 G. The results are shown in Figure 10. The simulated spectrum clearly captures the features of both the derivative and second harmonic representations. The multiplet reveals eight lines, although the outermost lines are very weak as is typical of these patterns.

Figure 10.

Expansion of the m_I = −1/2 lines of the S-band EPR spectra. The octarepeat (PHGGGWGQ) and HGGGW lines are shown (left) along with their derivatives (right). The simulated spectrum is from a model of three equivalent nitrogens (a_N = 13 G) and one proton (a_H = 10 G).

The multiplet pattern observed for the copper m_I = −1/2 component with a_N values typical of directly bound nitrogens strongly supports the suggestion of the previous section that several nitrogens participate in the component 1 Cu²⁺ coordination environment. Moreover, the values for g_‖, A_‖, and the superhyperfine couplings obtained at S-band are all readily explained by a coordination site consisting of three nitrogens. There still remains the possibility that only two nitrogens coordinate to Cu²⁺, and this would probably require unequal a_N values to explain the multiplet patterns in Figure 10. Future work with isotopic labeling, for example, will help resolve this issue and may also help identify the hydrogen that gives rise to the additional splitting. Finally, several attempts were made to obtain superhyperfine couplings from S-band spectra of PrP(23–28, 57–91). In all cases, no couplings were observed. The m_I = −1/2 line was slightly broader than that obtained from the octarepeat and HGGGW, suggesting that inhomogeneous broadening was masking the desired multiplet pattern. Fully loaded PrP(23–28, 57–91) contains four Cu²⁺ ions (Figures 5 and 6), and, hence, weak dipolar interactions between the paramagnetic centers may contribute an additional source of inhomogeneous broadening that is not canceled in the m_I = −1/2 line at S-band.

DISCUSSION

The EPR spectra in Figure 3 are all composed of two spectral components: component 1, which we propose arises from coordination of three nitrogens and one oxygen, and component 2, which may arise from two nitrogens and two oxygens. Component 1 dominates all of the spectra from peptides equal to or longer than the octarepeat, although component 2 is clearly present in several of the spectra. The simple sequence HGGGW gives rise to an almost pure component 1 spectrum. Given the sensitivity of EPR to coordination environment (e.g., symmetry, charge, and coordinating atoms), it appears that HGGGW binds Cu²⁺ in a fashion that is nearly equivalent to the multi-octarepeat peptides and therefore comprises the fundamental binding unit. Thus, we propose that fully bound PrP(57–91) takes up four Cu²⁺ ions with each metal ion coordinated to the HGGGW unit within an octarepeat. Interestingly, the peptides lacking a residue following the HGGG segment and also the shortest peptide HGG give rise to both component 1 and 2 spectra but now with a greater population of the latter. Thus, HGG may provide the necessary coordinating nitrogen atoms and the remainder of the sequence out to HGGGW and longer controls the equilibrium between two nitrogen and three nitrogen coordination.

The pH titrations, magnetic parameters g_‖ and A_‖, ¹⁴N superhyperfine couplings (from S-band EPR), and spectra from short peptides such as HGG argue that component 1 arises from Cu²⁺ coordination by three nitrogens. ESEEM demonstrates that the histidine imidazole contributes one of these nitrogens. The remaining two nitrogens must come from deprotonated backbone amide groups within the fundamental HGGGW unit. Comparable binding motifs for peptide–Cu²⁺ complexes are well-known (41, 42). Given the similarity of both component 1 and component 2 spectra across the peptide series it is worthwhile considering the uniqueness of Cu²⁺ EPR spectra. Specifically, is it possible for HGGGW and PrP(57–91), for example, to bind Cu²⁺ with the same number of nitrogens and oxygens but with participation of different residues? A recent study on tri- and tetrapeptides partially addresses this issue. Pogni et al. examined EPR of the series HGG, GHG, and GGH (43). While all of these peptides bound through either three or four nitrogens depending on pH, they gave vastly different EPR spectra especially in the g_⊥ region. Apparently, placement of the His within the sequence greatly influences the coordination site symmetry which is in turn reflected in the relative values of g_xx and g_yy. Thus, we conclude that HGGGW binds Cu²⁺ with a symmetry and ligand field that is equivalent to that obtained from the longer PrP peptides.

That each copper ion is bound mainly by residues within an octarepeat is further supported by titration experiments on the 1, 2, and 4 octarepeat containing peptides (Figures 5 and 6) where both EPR and CD demonstrate that the Cu²⁺/octarepeat stoichiometry is 1:1. This finding is consistent with the results of other researchers (11, 14, 16–18) with the exception of Viles et al. who report that PrP(51–75) and PrP(73–91), both of which contain two octarepeat units, bind only one copper ion (17). Our results with PrP(23–28,73–91) demonstrate binding to 2 equiv of Cu²⁺, and we are unable to account for the discrepancy between our data and that from Viles et al.

The EPR spectra (Figure 3) and spin integration (Figure 6, y axis) also address the possibility of imidazolate bridging between copper ions. To account for cooperative binding in PrP/Cu²⁺ complexes, Viles et al. proposed a novel ring structure for PrP(57–91) composed of four histidine residues bridging between four copper ions. Imidazolate bridging is well-documented in biological systems, such as Cu₂Zn₂ superoxide dismutase (44, 45) and in numerous model complexes (46, 47). An imidazolate bridge between a single pair of Cu²⁺ ions leads to an exchange interaction and, for typical strong coupling, the resulting EPR transitions take place solely among the two-spin triplet levels. If, in addition, the exchange interaction is large as compared to kT, the Boltzmann factor will favor the singlet ground state thereby decreasing the integrated EPR absorption signal relative to that expected from uncoupled spins. In the EPR monitored titration experiments performed on PrP(23–28, 57–91) at 298 K (Figure 6) the Cu²⁺/octarepeat stoichiometry was found to be approximately 1:1 when determined from spin integration. Additional spin quantitation was performed on fully Cu²⁺ loaded PrP(23–28, 57–91) as a function of temperature down to 77 K (data not shown), and the calibrated signal integrals revealed no variation to within 2% random error. Hence, there is no experimental evidence of signal loss due to exchange coupling. Typical values for the exchange interaction between Cu²⁺ pairs ranges from 17 to 90 cm⁻¹ (46), and such couplings will reduce the integrated signal intensity by 10 to 70%, respectively, at 77 K. We estimate that the largest possible error in signal integral is 15%, which may mask couplings as weak as 25 cm⁻¹. (Four coupled Cu²⁺ ions will lead to a comparable loss of integrated signal, but the picture is somewhat more complex than that described above.) Another diagnostic for coupling through imidazolates, however, is the EPR spectrum itself. For coupled species, the EPR signal is broad and, hence, quite distinct from mononuclear type 2 spectra such as those in Figure 3 (46–48). This unusual signal is also typically accompanied by a ΔM = 2 half field transition (46–48). As noted in Results, however, we did not observe a half field transition, and the EPR spectra displayed in Figure 3 are characteristic of isolated Cu²⁺ ions and show no unusual broadening or dipolar splittings as would be expected for a coupled species. These combined observations suggest that there is no EPR evidence in support of an imidazolate bridged species in PrP(57–91) when bound to four Cu²⁺ ions.

In a recent Raman study of octarepeat-derived peptides, Miura et al. concluded that the segment HGGG is the fundamental Cu²⁺ binding unit at pH 7.4 (16, 18). Further, their study indicated that the peptide coordinates Cu²⁺ via the N_π atom (also denoted Nδ1) of the histidine side chain and two deprotonated main chain amide nitrogens. Our findings are largely consistent with theirs, but we find evidence for two binding modes within the octarepeat. Further, a Trp residue following the HGGG binding unit is needed to reproduce the dominant spectral component of the multi-octarepeat peptides.

Together these data indicate that the majority of copper is bound within the segment HGGG and that each octarepeat segment binds copper independently. Component 1 in the EPR spectra of Figure 3 therefore arises from coordination of the histidine side chain nitrogen and two deprotonated glycine amide nitrogens. Component 2 may then arise from a state in which one of the glycine amides has released Cu²⁺ and been replaced by an oxygen-containing ligand such as a backbone carbonyl or water. The presence of the tryptophan (or X) residue after the HGGG segment clearly plays a role in the equilibrium between these two binding modes.

Figure 11 shows our working model for the dominant (component 1) octarepeat binding mode. EPR is mainly sensitive to the equatorial coordination shown in the model, but we recognize that there are probably oxygens from water or hydroxide at the axial positions. Because the EPR spectrum of copper bound HGG still gives a component 1 spectrum, although reduced in intensity, we propose that along with the imidazole, the next two glycine amides participate in Cu²⁺ coordination. Crystal structures of peptide–Cu²⁺ complexes indeed show that coordination of sequential backbone amides is common (41, 42, 49). The EPR data also suggest that a residue following the HGGG segment (W or X) is necessary to give a dominant component 1 spectra, and we propose that this additional residue contributes an oxygen to the coordination environment. Thus, in our model, the Trp carbonyl is shown coordinating the metal ion center. Not only is this model consistent with our findings, but it also explains the near proximity of the Trp indole to the paramagnetic center demonstrated by fluorescence quenching experiments (15). Miura et al. have also proposed coordination by two amides (18), and our model is also consistent with their experimental data. Finally, we note that a crankshaft rearrangement of one of the first or second Gly to replace a coordinated amide nitrogen with a carbonyl may explain the component 2 spectrum that dominates at lower pH.

Figure 11.

Proposed bond-line model of the dominant binding mode (component 1) of the octarepeat segment (PHGGGWGQ).

For a fully Cu²⁺ loaded protein, this model suggests that the metal ion binding sites in the N-terminal domain are like beads on a string where each bead is a Cu²⁺-HGGGW segment separated by intervening Gly–Gln–Pro (GQP) links. Interestingly, Gly and Pro often participate in β-turns, and thus the intervening links may provide a mechanism for allowing the Cu²⁺ binding segments to fold and perhaps collapse together. If the model in Figure 11 proves to be correct, it will represent a novel Cu²⁺ binding site in proteins. Although serum albumin binds Cu²⁺ in a GGH segment via amide nitrogens (50), most copper binding proteins coordinate the metal ion through side chain functionalities (e.g., His, Cys, Met). Moreover, the proposed binding mode may figure nicely into current theories of PrP function. Cu²⁺ stimulates endocytosis (13), and it is proposed that PrP’s role may be to sense the concentration of Cu²⁺ and shuttle the metal ion from the extracellular domain at pH > 7 to endo-lysosomes at reduced pH (19, 25). To support this hypothesis, PrP must exhibit pH-sensitive binding. Amide coordination to Cu²⁺ as shown in Figure 11 is extremely pH-sensitive. In the absence of Cu²⁺, backbone amide pK_a’s are in the range of 13 to 15. However, when Cu²⁺ is brought into proximity by coordination to a His side chain, the pK_a’s of nearby amides are greatly reduced and often below 7 (51). At pH > 7 these amides readily deprotonate and coordinate Cu²⁺ through the nitrogen. However, if the pH is lowered by one or two units, protonation competes with Cu²⁺ coordination thereby lowering the affinity for the metal ion (52, 53). Figures 1 and 2 clearly reflect substantial pH sensitivity in the PrP octarepeat domain. In addition, ESI-MS of PrP(57–91) suggests that only two Cu²⁺ bind at pH 6 (19). The model proposed in Figure 11 accounts for differential Cu²⁺ affinity above and below pH = 7 and is consistent with current theories linking PrP function to Cu²⁺ shuttling through endocytosis.

CONCLUSION

These studies combined peptide design with EPR, ESEEM, CD spectroscopy, and titration experiments to evaluate the Cu²⁺ binding unit of the PrP N-terminus. The findings help resolve whether each Cu²⁺ binds within a single octarepeat (15, 16, 18) or between octarepeats via imidazolate bridging (17). The EPR studies further show that there are two binding modes at pH 7.4 where the dominant mode arises from coordination of three nitrogens. In addition, the way in which Cu²⁺ binds to the multi-octrapeat peptides appears to depend on the relative metal ion and peptide concentrations. The simple peptide unit HGGGW possesses the necessary features to bind Cu²⁺ in a manner that is spectroscopically indistinguishable from the dominant binding mode observed for the multi-octarepeat peptides. Our observations are collected into a working model for binding above pH 7 where we propose that coordination in the equatorial plane arises from a nitrogen of the His imidazole, two backbone nitrogens from the following glycines, and the amide carbonyl of the tryptophan. Although this model needs further scrutiny, it provides a starting point for evaluating the metal ion induced structure of the PrP N-terminus and the mechanism by which PrP takes up and releases Cu²⁺. Future work will also focus on elucidation of the component 2 binding mode, its possible physiological role, and how low pH leads to specific changes in the Cu²⁺ binding environment.