Methionine oxidation perturbs the structural core of the prion protein and suggests a generic misfolding pathway

Abstract

Oxidative stress and misfolding of the prion protein (PrP(C)) are fundamental to prion diseases. We have therefore probed the effect of oxidation on the structure and stability of PrP(C). Urea unfolding studies indicate that H(2)O(2) oxidation reduces the thermodynamic stability of PrP(C) by as much as 9 kJ/mol. (1)H-(15)N NMR studies indicate methionine oxidation perturbs key hydrophobic residues on one face of helix-C as follows: Met-205, Val-209, and Met-212 together with residues Val-160 and Tyr-156. These hydrophobic residues pack together and form the structured core of the protein, stabilizing its ternary structure. Copper-catalyzed oxidation of PrP(C) causes a more significant alteration of the structure, generating a monomeric molten globule species that retains its native helical content. Further copper-catalyzed oxidation promotes extended β-strand structures that lack a cooperative fold. This transition from the helical molten globule to β-conformation has striking similarities to a misfolding intermediate generated at low pH. PrP may therefore share a generic misfolding pathway to amyloid fibers, irrespective of the conditions promoting misfolding. Our observations support the hypothesis that oxidation of PrP destabilizes the native fold of PrP(C), facilitating the transition to PrP(Sc). This study gives a structural and thermodynamic explanation for the high levels of oxidized methionine in scrapie isolates.

FIGURE 1.

Structural changes upon methionine oxidation of PrP indicated by chemical shift perturbations. a, two-dimensional ¹H-¹⁵N HSQC spectra of PrP(113–231) (3.0 mg/ml) in 10 mm acetate buffer, pH 5.6, at 37 °C, showing unoxidized spectra (black) and H₂O₂ (10 mm)-incubated sample after 3 h (pink) and 9 h (blue) of incubation. b, plot of chemical shift perturbation per residue. Strongly perturbed amide resonances are highlighted along with a representation of the secondary structural regions of PrP^C showing the three α-helices (residues 144–156, 172–193, and 200–227) and two short anti-parallel β-strands (128–131 and 161–164). c, structure of mouse PrP^C (chemical shift perturbation code 1XYX) with key perturbed side chains shown.

FIGURE 2.

Urea unfolding of H₂O₂-oxidized PrP(23–231). CD data at 225 nm with increasing [urea] for unoxidized PrP(23–231) (triangles), H₂O₂-oxidized PrP(23–231) under mild condition (circles), repeat measurements (crosses), and H₂O₂-oxidized PrP(23–231) under harsher conditions (squares) are shown. All PrP samples (130 μm) were incubated with 10 or 300 mm H₂O₂ for 8 h before urea titration. The protein concentration was diluted to 4.3 μm, pH 5.5, using 10 mm sodium acetate buffer.

FIGURE 3.

Copper-catalyzed oxidation of PrP(23–231). a, unoxidized; b, after 4 h of Cu²⁺ + H₂O₂ oxidized; c, 16 h after copper-catalyzed oxidation. After 4 h some resonances from helix-C retain their signal and chemical shift values (labeled in black), although many other signals from the structured domain lose their signal intensity (labeled in gray) due to exchange broadening of a molten globule fold. After 16 h only signals from the N-terminal residues (23–122) were observed. 130 μm PrP(23–231) was used with 0.1 mol eq of Cu²⁺ ions with 10 mm H₂O₂ in 10 mm sodium acetate buffer, pH 5.6.

FIGURE 4.

Size-exclusion chromatogram of Cu²⁺-catalyzed oxidation of PrP(23–231). A series of chromatograms showing the change in Cu²⁺-catalyzed oxidation of PrP(23–231) under the milder conditions, from unoxidized and freshly oxidized to 27 h of incubation at 37 °C. All were carried out using a Superdex-75 column. The incubation was carried out using 130 μm PrP(23–231) with 0.1 mol eq of Cu²⁺ ions and 10 mm H₂O₂ in 10 mm sodium acetate buffer, pH 5.5.

FIGURE 5.

ANS (a) and bis-ANS (b) fluorescence for oxidized H₂O₂ and copper-oxidized PrP. 130 μm PrP(23–231) was oxidized by incubation with 10 mm H₂O₂ (black line) or with 10 mm H₂O₂ plus 0.1 mol eq Cu²⁺ ions (gray line). Samples were incubated for 22 h at 37 °C, before being diluted to 4 μm for fluorescence assay. Unoxidized PrP(23–231) is also shown. Under this relatively mild oxidizing condition, both H₂O₂ and copper-catalyzed oxidations caused increased fluorescence relative to unoxidized PrP(23–231) for both ANS and bis-ANS binding suggesting a molten globule folding intermediate had formed.

FIGURE 6.

UV-CD of oxidation of PrP. Structural transitions monitored by UV-CD of full-length and a fragment of PrP when incubated with H₂O₂, with and without the presence of Cu²⁺ ions. a, PrP(23–231) (130 μm) incubated with H₂O₂ (10 mm) and Cu²⁺ (0.1 mol eq); b, PrP(23–231) (130 mm) incubated with H₂O₂ (10 mm) only; c, PrP(113–231) (4 μm) and Cu²⁺ (0.1 mol eq) incubated with H₂O₂ (10 mm), spectra at 2-h time intervals; d, PrP(113–231) (4 μm) incubated with H₂O₂ (10 mm) only; e, PrP(23–231) (4 μm) and Cu²⁺ (0.1 mol eq) incubated with H₂O₂ (10 mm), spectra at 2-h time intervals, are shown. f, PrP(23–231) (4 μm) incubated with H₂O₂ (10 mm) only. All spectra recorded at pH 5.5 at 37 °C and diluted to 4 μm PrP.

FIGURE 7.

Urea unfolding of Cu²⁺-catalyzed oxidized PrP(23–231). CD data at 225 nm with increasing [urea] of unoxidized PrP(23–231) (triangles), PrP(23–231) with 0.1 mol eq of Cu²⁺ (filled triangles), Cu²⁺-catalyzed oxidized PrP(23–231) under the mild conditions (circles), and Cu²⁺-catalyzed oxidized PrP(23–231) under harsher conditions (squares) are shown. All PrP samples (130 μm) were incubated with 0.1 mol eq Cu²⁺ and 10 or 300 mm H₂O₂ for 8 h before urea titration. The protein concentration was diluted to 4.3 μm, pH 5.5, using 10 mm sodium acetate buffer.

FIGURE 8.

FT-IR spectra of full-length PrPC oxidized. a, copper-catalyzed; b, H₂O₂ only. Amide-I band of unoxidized PrP(23–231) (black line) and oxidized PrP(23–231) (gray line). Oxidized PrP was generated by incubating for 20 h under conditions described in Fig. 6, e and f. The oxidized PrP(23–231) contains an amide-I band at 1630 cm⁻¹ diagnostic of an increase in β-sheet content, particularly apparent for the Cu²⁺-catalyzed oxidized PrP.

FIGURE 9.

Comparison of PrP(23–231) generated at pH 4 to Cu²⁺-catalyzed oxidation of PrP(23–231). Two-dimensional ¹H-¹⁵N HSQC spectra of PrP(23–231) (5.0 mg/ml) in 150 mm NaCl, 20 mm sodium acetate buffer, pH 3.6, 37 °C, in black and PrP(23–231) (3.0 mg/ml) in 10 mm sodium acetate buffer, pH 5.6, in the presence of H₂O₂ (10 mm) and 0.1 mol eq Cu²⁺ ions after 16 h of incubation at 37 °C in red.

FIGURE 10.

Misfolding pathway of PrP upon oxidation. Oxidation of methionines destabilizes the hydrophobic core of PrP^C, and wider copper-catalyzed oxidation generates a monomeric molten globule of PrP with a high helical content. Further oxidation generates an extended β-conformation that lacks a stable cooperative fold. This has striking parallels with the pH 4 intermediate (see Table 3), which will go on and form amyloid fibers.