Conformational pH dependence of intermediate states during oligomerization of the human prion protein

Abstract

Intermediate states are key to understanding the molecular mechanisms governing protein misfolding. The human prion protein (PrP) can follow various misfolding pathways, and forms a soluble beta-sheet-rich oligomer under acidic, mildly denaturing, high salt conditions. Here we describe a fast conformational switch from the native alpha-monomer to monomeric intermediate states under oligomer-forming conditions, followed by a slower oligomerization process. We observe a pH dependence of the secondary structure of these intermediate forms, with almost native-like alpha-helical secondary structure at pH 4.1 and predominantly beta-sheet characteristics at pH 3.6. NMR spectroscopy differentiates these intermediate states from the native protein and indicates dynamic rearrangements of secondary structure elements characteristic of a molten globule. The alpha-helical intermediate formed at pH 4.1 can convert to the beta-sheet conformation at pH 3.6 but not vice versa, and neither state can be reconverted to an alpha-monomer. The presence of methionine rather than valine at codon 129 accelerates the rate of oligomer formation from the intermediate state.

Figure 1.

Misfolding model of human prion protein in vitro. In this schematic, the various in vitro folding pathways of recombinant, disulfide-oxidized PrP are summarized as discussed in the literature (Baskakov et al. 2001, 2002; Tahiri-Alaoui et al. 2006). It was recently shown that the intermediate state along pathway 1 is more populated during folding at low pH 4.8 in comparison to folding at pH 7.0 (Apetri et al. 2006). The shaded area of intermediate states during misfolding is of particular interest due to the unknown molecular mechanism governing and initiating prion misfolding. In this work we differentiate the intermediate folding landscape by showing the existence of α-helical and β-sheet-rich monomeric precursors αⁱ and βⁱ during misfolding along pathway 6. The effects on the folding kinetics are shown with respect to residue 129 of the common polymorphism encoding either methionine (M) or valine (V) or for their equimolar mixture (MV) (Tahiri-Alaoui et al. 2004, 2006; Baskakov et al. 2005; Tahiri-Alaoui and James 2005). Here we report an influence of codon 129 on pathway 6. The bold dotted lines indicate that the formation of β-oligomer proceeds through two different pathways that are most likely separated by a free energy barrier.

Figure 2.

Identification of monomeric folding intermediates with either α-helical or β-sheet secondary structures depending on pH. (A,B) Oligomerization of PrP was monitored with far-UV CD at pH 4.1 and pH 3.6, respectively. While the spectra of α^N and β^O were recorded for the stable species, the evolving αⁱ and βⁱ were monitored at incubation times of 5, 45, 90, 180, and 240 min and 53 h, as indicated. (C) The sec-HPLC chromatograms for α^N, β^O, αⁱ (dashed line) and βⁱ (dotted-dashed line) eluting at 9.89, 6.58, 9.11, and 9.44 min are shown, respectively. The elution profiles of α^N, αⁱ, and βⁱ are distinguishable by their elution times between 9 and 10 min.

Figure 3.

Oligomerization intermediates have nonnative tertiary structure. (A–C) The ¹H-¹⁵N HSQC spectra of α^N, αⁱ, and βⁱ are shown, respectively. Resonances in A were assigned according to Hosszu et al. (2004), and peaks that are observed in all three spectra are marked with an asterisk. (B,C) The HSQC spectra of both αⁱ and βⁱ show about 60 sharp resonances, most of which fall within the random-coil region between 7.7 and 8.7 ppm ¹H chemical shift. The loss of dispersed resonances is indicative of a nonnative molten globule conformation.

Figure 4.

The intermediate states αⁱ and βⁱ cannot be reversed to α-monomer. (A) Native α^N was converted to αⁱ and then the pH was either dropped to pH 3.6 converting αⁱ to βⁱ or raised to pH 5.5. (B) Similar to A, α^N was converted into βⁱ, and subsequently, the pH was raised back to either pH 4.1 or pH 5.5. (C) The ¹H-¹⁵N HSQC spectrum of βⁱ after the pH had been shifted back to pH 5.5 shows a near complete loss of sharp resonances. (D) A schematic of the pathways accessible to αⁱ and βⁱ as probed with CD is shown.

Figure 5.

The late stage of oligomerization on pathway 6 is affected by codon 129. Pathway 6 misfolding was monitored by injecting samples onto a sec-HPLC column at different incubation times under oligomer-favoring conditions. Oligomerization of Met¹²⁹ (A,D), Val¹²⁹ (B,E), and the equimolar mixture Met/Val¹²⁹ (C,F) was monitored at both pH 4.1 (A–C) and pH 3.6 (D–E), respectively. The β^M (multimer as described by Baskakov et al. [2001, 2002, 2005]) β^O, αⁱ, and βⁱ fractions eluting at 5.0, 6.58, 9.44, and 9.11 min are indicated in the figure (A and D), respectively. (G,H) The non-monomeric fractions of 5.0–8.7 min of the elution profiles were integrated and mono-exponential association curves fitted to the points for Met¹²⁹ (■), Val¹²⁹ (▼), Met/Val¹²⁹ (●), respectively. (I) The rate constants for Met¹²⁹, Val¹²⁹, and Met/Val¹²⁹ indicate the most rapid oligomerization for Met¹²⁹. The rates for pH 3.6 and pH 4.1 are within the same order but plateau levels are overall lower at pH 4.1.