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

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.

Inter-oligomer interactions of the human prion protein are modulated by the polymorphism at codon 129.

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in beta-oligomer (beta(O)) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the beta(O) N-terminal segments, showing that it is the core region of PrP (residues 127-228) that is involved in beta(O) formation. Atomic force microscopy revealed distinctive 1.8 x 15 x 15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for beta(O) with valine at position 129. This result provides evidence for conformational differences between the beta(O) allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.

Oligomerization of the human prion protein proceeds via a molten globule intermediate.

The conformational transition of the human prion protein from an alpha-helical to a beta-sheet-rich structure is believed to be the critical event in prion pathogenesis. The molecular mechanism of misfolding and the role of intermediate states during this transition remain poorly understood. To overcome the obstacle of insolubility of amyloid fibrils, we have studied a beta-sheet-rich misfolded isoform of the prion protein, the beta-oligomer, which shares some structural properties with amyloid, including partial proteinase resistance. We demonstrate here that the beta-oligomer can be studied by solution-state NMR spectroscopy and obtain insights into the misfolding mechanism via its transient monomeric precursor. It is often assumed that misfolding into beta-sheet-rich isoforms proceeds via a compatible precursor with a beta-sheet subunit structure. We show here, on the contrary, evidence for an almost natively alpha-helix-rich monomeric precursor state with molten globule characteristics, converting in vitro into the beta-oligomer. We propose a possible mechanism for the formation of the beta-oligomer, triggered by intermolecular contacts between constantly rearranging structures. It is concluded that the beta-oligomer is not preceded by precursors with beta-sheet structure but by a partially unfolded clearly distinguishable alpha-helical state.