Copper binding to the prion protein: structural implications of four identical cooperative binding sites

Abstract

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd approximately 6 microM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nepsilon2 and Ndelta1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.

Figure 1

(A) UV CD spectrum (180–260 nm) of PrP(58–91) (0.032 mM, pH 7.5, no buffer) with the addition of Cu(II) in increments of 0.33 mole-equivalents of CuSO₄ from 0 (light blue) up to 7 mole-equivalents (orange). (B) Visible CD spectrum (300–750 nm) of PrP(58–91) (0.033 mM, pH 7.5, no buffer) with the addition of Cu(II) in increments of 1.0 mole-equivalent of CuSO₄ from 0 (light blue) up to 6 mole-equivalents (orange). The curves for 1, 3, and 7 mole-equivalents are represented as single points at 570 nm.

Figure 2

(A) Cu(II) binding curves: molar ellipticity at 570 nm with increasing amounts of Cu(II), pH 7.5. ●, 2-His peptide, PrP(51–75) (0.34 mM). ⧫, 3-His peptide, PrP(66–91) (0.021 mM). ▴, 4-His peptide, PrP(58–91) (0.033 mM). (B) pH dependence of the ellipticity at 570 nm for PrP(58–91). The pH dependence curve has been fitted to the following equation: Δɛ_obs = {Δɛ_acid[H⁺]ⁿ + Δɛ_base[H⁺] K_aⁿ }/ [H⁺]ⁿ + K_aⁿ}, where n = Hill coefficient and K_a = acid dissociation constant for the transition. The midpoint of the transition is pH 6.7.

Figure 3

X-band ESR spectra of 1, 2, and 3 mole-equivalents of Cu(II) added to PrP(58–91). A spectrum of 0.8 mole-equivalents of Cu(II) added to PrP(73–91) also is shown. Samples were buffered with 10 mM N-ethylmorpholine HCl, and spectra were acquired at 5 K.

Figure 4

¹H NMR stack plot showing the effects of addition of Cu(II) to PrP(76–86) (0.5 mM in 90/10% H₂O/D₂O, pH 7.4, and 10 mM phosphate buffer at 10°C). The CuSO₄ was added in aliquots of 0.0033 mole-equivalents up to 0.02 mole-equivalents. The spectral width for each panel is 150 Hz.

Figure 5

CD spectrum (300–750 nm) of full length PrP(29–231) after the addition of 25 mole-equivalents of Cu(II) at pH 5.3, 6.2, and 6.5 in N-ethylmorpholine buffer.

Figure 6

(A) A plausible structure for the complex of Cu(II) with PrP(76–86). (B) A plausible structure for the bridged complex of four Cu(II) ions with PrP(58–91).