Molecular features of the copper binding sites in the octarepeat domain of the prion protein

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. 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 Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximately 6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.     

Copper binding to the octarepeats of the prion protein. Affinity,specificity, folding,and cooperativity: insights from circular dichroism

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein. There is increasing evidence that PrP functions as a copper transporter. In addition, strains of prion disease have been linked with copper binding. We present here CD spectroscopic studies of Cu(2+) binding to various fragments of the octarepeat region of the prion protein. We show that glycine and l-histidine will successfully compete for all Cu(2+) ions bound to the PrP octapeptide region, suggesting Cu(2+) coordinates with a lower affinity for PrP than the fm dissociation constant reported previously. We show that each of the octarepeats do not form an isolated Cu(2+) binding motif but fold up cooperatively within multiple repeats. In addition to the coordinating histidine side chain residues, we show that the glycine residues and the proline within each octarepeat are also necessary to maintain the coordination geometry. The highly conserved octarepeat region in mammals is a hexarepeat in birds that also binds copper but with different coordination geometry. Finally, in contrast to other reports, we show that Mn(2+) does not bind to the octarepeat region of PrP.     

Deconvoluting the Cu2+ binding modes of full-length prion protein

The prion protein (PrP) is a cell-surface Cu(2+)-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23-231) and constructs in which the octarepeat region has been removed, or His(95) and His(110) is replaced by alanine residues, have been used to elucidate the order and mode of Cu(2+) coordination to PrP-(23-231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu(2+) coordination to full-length PrP. At physiological pH, Cu(2+) initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Ni(2+) ions are used to further probe metal binding and, like Cu(2+), Ni(2+) will bind individually to His(95) and His(110), involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+) binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.     

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

The prion protein (PrP) binds Cu(2+) in its N-terminal octarepeat domain, composed of four or more tandem PHGGGWGQ segments. Previous work from our laboratory demonstrates that copper interacts with the octarepeat domain through three distinct coordination modes at pH 7.4, depending upon the precise ratio of Cu(2+) to protein. Here, we apply both electron paramagnetic resonance (EPR) and fluorescence quenching to determine the copper affinity for each of these modes. At low copper occupancy, which favors multiple His coordination, the octarepeat domain binds Cu(2+) with a dissociation constant of 0.10 (+/-0.08) nM. In contrast, high copper occupancy, involving coordination through deprotonated amide nitrogens, exhibits a weaker affinity characterized by dissociation constants in the range of 7.0-12.0 microM. Decomposition of the EPR spectra reveals the proportions of all coordination species throughout the copper concentration range and identifies significant populations of intermediates, consistent with negative cooperativity. At most copper concentrations, the Hill coefficient is less than 1.0 and approximately 0.7 at half copper occupancy. These findings demonstrate that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude. Consideration of these findings, along with the demonstrated ability of the protein to quench copper redox activity at high occupancy, suggests that PrP may function to protect cells by scavenging excess copper.     

Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.     

Copper coordination in the full-length,recombinant prion protein

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment constitutes the fundamental binding unit in the octarepeat domain [Burns et al. (2002) Biochemistry 41, 3991-4001; Aronoff-Spencer et al. (2000) Biochemistry 39, 13760-13771]. Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.     

Computational studies of Cu(II)[peptide] binding motifs: Cu[HGGG] and Cu[HG] as models for Cu(II) binding to the prion protein octarepeat region

The binding of Cu(II) to the prion protein is investigated by computations at the B3LYP level of theory on models of the octarepeat domain of the prion protein. The models incorporate the functionality of the glycine (G) and histidine (H) residues which occur in the octarepeat domain, PHGGGWGQ. The copper complexes are designated Cu[HG] and Cu[HGGG]. Coordination to the metal via the imidazole ring of the histidine, the amide carbonyl groups, and the backbone nitrogen atom of the amide groups were examined, as well as several protonation/deprotonation states of each structure. EPR and CD titration experiments suggest that the octarepeat segments of the unstructured N-terminal domain of prion protein can bind Cu(II) in a 1:1 Cu-to-octarepeat ratio. The results identify the extent to which the Cu(II) facilitates peptide backbone deprotonation, and the propensity of binding in the forward (toward the C-terminus) direction from the anchoring histidine residue. A plausible mechanism is suggested for changing from amide O-atom to deprotonated amide N-atom coordination, and for assembly of the observed species in solutions of Cu[PrP] and truncated models of it. A structure is proposed which has the N2O2 coordination pattern for the minor component observed experimentally by EPR spectroscopy for the Cu[HGGG] model. The most stable neutral Cu[HGGG] structure found, with coordination environment N3O1, corresponds to that observed for Cu[HGGGW] and Cu[HGGG] both in the solid state and as the major component in solution at neutral pH.     

Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.     

Copper(II) complexes of peptide fragments of the prion protein. Conformation changes induced by copper(II) and the binding motif in C-terminal protein region

In this paper, we report the characterization of copper(II) complexes with two prion (PrP) protein peptide fragment analogues (VNITKQHTVTTTT), one with the N-terminus acetylated and the C-terminus amidated (PrP Ac180-193NH2) and the other with both the C- and N-termini free (PrP 180-193). Such peptide sequence almost entirely encompasses the PrPC's helix 2 in the C-terminal region. The stoichiometry, the binding modes and the conformational features of the copper(II) complexes with the above mentioned two peptides were investigated by electrospray ionization-mass spectrometry (ESI-MS), UV-visible (UV-Vis) spectrometry and electron paramagnetic resonance (EPR) spectrometry as well as by circular dichroism (CD) measurements. The binding site location of copper(II) in the structured region of the protein can be here suggested on the basis of our findings that show the involvement of His 187 residue. The similarity of the EPR parameters suggests that the anchoring imidazole residue drives the copper(II) coordination environment towards a common binding motif in different regions of the prion protein.     

The configuration of the Cu2+ binding region in full-length human prion protein

The cellular prion protein (PrP^C) is a Cu²⁺ binding protein connected to the outer cell membrane. The molecular features of the Cu²⁺ binding sites have been investigated and characterized by spectroscopic experiments on PrP^C-derived peptides and the recombinant human full-length PrP^C (hPrP-[23-231]). The hPrP-[23-231] was loaded with ⁶³Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP^C/Cu²⁺-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu²⁺ structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu²⁺ coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP^C. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP(C)). The precise physiological function of PrP(C) remains uncertain; however, Cu(2+) binds to PrP(C) in vivo, suggesting a role for PrP(C) in copper homeostasis. Here we examine the oxidative processes associated with PrP(C) and Cu(2+). (1)H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl(2) showed specific metal-catalyzed oxidation of histidine residues, His(96/111), and the methionine residues, Met(109/112). The octarepeat region protects His(96/111) and Met(109/112) from oxidation, suggesting that PrP(90-231) might be more prone to chemical modification. We show that Cu(2+/+) redox cycling is not 'silenced' by Cu(2+) binding to PrP, as indicated by H(2)O(2) production for full-length PrP. Surprisingly, although detection of Cu(+) indicates that the octarepeat region of PrP is capable of reducing Cu(2+) even in the absence of ascorbate, H(2)O(2) is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu(2+)/O(2) system; however, levels of H(2)O(2) production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP(C).     

A spectroscopic and voltammetric study of the pH-dependent Cu(II) coordination to the peptide GGGTH: relevance to the fifth Cu(II) site in the prion protein

The GGGTH sequence has been proposed to be the minimal sequence involved in the binding of a fifth Cu(II) ion in addition to the octarepeat region of the prion protein (PrP) which binds four Cu(II) ions. Coordination of Cu(II) by the N- and C-protected Ac-GGGTH-NH(2) pentapeptide (P(5)) was investigated by using potentiometric titration, electrospray ionization mass spectrometry, UV-vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry experiments. Four different Cu(II) complexes were identified and characterized as a function of pH. The Cu(II) binding mode switches from NO(3) to N(4) for pH values ranging from 6.0 to 10.0. Quasi-reversible reduction of the [Cu(II)(P(5))H(-2)] complex formed at pH 6.7 occurs at E (1/2)=0.04 V versus Ag/AgCl, whereas reversible oxidation of the [Cu(II)(P(5))H(-3)](-) complex formed at pH 10.0 occurs at E (1/2)=0.66 V versus Ag/AgCl. Comparison of our EPR data with those of the rSHaPrP(90-231) (Burns et al. in Biochemistry 42:6794-6803, 2003) strongly suggests an N(3)O binding mode at physiological pH for the fifth Cu(II) site in the protein.     

Empirical rules for rationalising visible circular dichroism of Cu2+ and Ni2+ histidine complexes: applications to the prion protein

A natively unfolded region of the prion protein, PrP(90-126) binds Cu(2+) ions and is vital for prion propagation. Pentapeptides, acyl-GGGTH(92-96) and acyl-TNMKH(107-111), represent the minimum motif for this Cu(2+) binding region. EPR and (1)H NMR suggests that the coordination geometry for the two binding sites is very similar. However, the visible CD spectra of the two sites are very different, producing almost mirror image spectra. We have used a series of analogues of the pentapeptides containing His(96) and His(111) to rationalise these differences in the visible CD spectra. Using simple histidine-containing tri-peptides we have formulated a set of empirical rules that can predict the appearance of Cu(2+) visible CD spectra involving histidine and amide main-chain coordination.     

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.     

Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein

The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60–91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91–230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).     

Identification of the copper(II) coordinating residues in the prion protein by metal-catalyzed oxidation mass spectrometry: evidence for multiple isomers at low copper(II) loadings

While the Cu(II) binding sites of the prion protein have been well studied under Cu-saturation conditions, the identity of the residues involved in coordinating Cu(II) at low stoichiometries and the order in which the binding sites load with Cu(II) remain unresolved. In this study, we have used two mass spectrometry based methods to gather insight into Cu(II)-prion binding under different stoichiometric loadings of Cu(II). The first method uses metal-catalyzed oxidation reactions to site specifically modify the residues bound to Cu(II) in solution, and the second method determines Cu binding sites based on the protection of His from modification by diethyl pyrocarbonate when this residue binds Cu(II) in solution. For both methods, the residues that are labeled by these reactions can then be unambiguously identified using tandem mass spectrometry. Upon applying these two complementary methods to a construct of the prion protein that contains residues 23-28 and 57-98, several noteworthy observations are made. Coordination of Cu(II) by multiple His imidazoles is found at 1:1 and 1:2 PrP:Cu(II) ratios. Notably, there appear to be four to seven isomers of this multiple histidine coordination mode in the 1:1 complex. Furthermore, our data clearly show that His96 is the dominant Cu(II) binding ligand, as in every isomer His96 is bound to Cu(II). The individual octarepeat binding sites begin to fill at ratios of 1:3 PrP:Cu(II) with no clear preference for the order in which they load with Cu(II), although the His77 octarepeat appears to saturate last. The existence of several "degenerate" Cu binding modes at low PrP:Cu(II) ratios may allow it to more readily accept additional Cu(II) ions, thus allowing PrP to transition from a singly Cu(II) bound state to a multiply Cu(II) bound state as a function of cellular Cu(II) concentration.     

The copper(II) binding properties of the cyclic peptide c(HGHK)

The new cyclic tetrapeptide c(HGHK) was synthesised in the solid phase and its complexes with copper(II) were studied in aqueous solution at various pH values by means of potentiometric and spectroscopic methods (UV, EPR, CD). Six mononuclear coordination species were clearly identified within the pH range 3-11. Spectroscopic data strongly suggest sequential formation of N, 2N, 3N and 4N equatorial donor sets around the copper(II) centre from the lowest to the highest pH, involving both imidazole nitrogens and amide nitrogens. A detailed comparison with the copper(II) binding properties of HGHG and Ac-HGHG ligands is also reported.     

Mapping Cu(II) binding sites in prion proteins by diethyl pyrocarbonate modification and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric footprinting.

Although Cu(II) ions bind to the prion protein (PrP), there have been conflicting findings concerning the number and location of binding sites. We have combined diethyl pyrocarbonate (DEPC)-mediated carbethoxylation, protease digestion, and mass spectrometric analysis of apo-PrP and copper-coordinated mouse PrP23-231 to "footprint" histidine-dependent Cu(II) coordination sites within this molecule. At pH 7.4 Cu(II) protected five histidine residues from DEPC modification. No protection was afforded by Ca(II), Mn(II), or Mg(II) ions, and only one or two residues were protected by Zn(II) or Ni(II) ions. Post-source decay mapping of DEPC-modified histidines pinpointed residues 60, 68, 76, and 84 within the four PHGGG/SWGQ octarepeat units and residue 95 within the related sequence GGGTHNQ. Besides defining a copper site within the protease-resistant core of PrP, our findings suggest application of DEPC footprinting methodologies to probe copper occupancy and pathogenesis-associated conformational changes in PrP purified from tissue samples.     

A reassessment of copper(II) binding in the full-length prion protein

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.