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. 2007 Nov 27;104(48):18946-51.
doi: 10.1073/pnas.0706522104. Epub 2007 Nov 19.

Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure

Nathan J Cobb  1 , Frank D Sönnichsen, Hassane McHaourab, Witold K Surewicz
Affiliations

Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure

Nathan J Cobb et al. Proc Natl Acad Sci U S A. .

Abstract

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and alpha-helical prion protein, PrP(C), to the beta-sheet-rich PrP(Sc). This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP(C) monomer, structures of the pathogenic PrP(Sc) or synthetic PrP(Sc)-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP(C). Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrP(C) involves major refolding of the C-terminal alpha-helical region. The core of the amyloid maps to C-terminal residues from approximately 160-220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of beta-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to beta-structure.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Spin exchange in prion amyloid fibrils. (A) EPR spectra for the representative huPrP90–231 variant spin labeled at position 188 in the monomeric (black line) and fibrillized (red line) forms. (B) EPR spectra for amyloid fibrils generated by using labeled protein alone (red line) and mixtures of labeled and unlabeled protein at a 1:1 ratio (black line) and 1:4 ratio (gray line). Spectra within each panel (but not between panels) are normalized to the same number of spins, and for visualization purposes, each panel is scaled to the same vertical size. Scan width is 150 G.
Fig. 2.
Fig. 2.
EPR signals obtained from fibrils generated by using D178N huPrP 90–231 labeled at indicated positions. For clarity of presentation, spectra displaying a high degree of spin exchange are shown in red, and those spectra affected largely by dipolar broadening are shown in black. Spectra for spin-diluted fibrils generated by using a 1:4 ratio of labeled to unlabeled protein are shown as gray lines. Scan width is 200 G, and spectral pairs for each site are scaled to the same number of spins.
Fig. 3.
Fig. 3.
Changes in central line amplitude for EPR spectra of fibrils resuspended in 1 and 2 M GdnHCl relative to the amplitude in the absence of denaturant. Spectra for sites ≈160–220 show little change, and thus the molecular architecture of this region is maintained in the presence of the denaturant. Residues N-terminal to 160 display particularly large increases in amplitude, indicating low stability of any structure in this region to chemical denaturation. Relatively small responses observed for the most N-terminal sites (97 and 103) are due to already large interspin distances and mobilities of these sites in the absence of denaturant (see Fig. 2).
Fig. 4.
Fig. 4.
The simplest model of recombinant PrP amyloid consistent with present experimental data. (A) The monomeric folded domain (residues 120–231) of human PrP. Residues determined herein to form the core of the amyloid are highlighted in red. (B) Electron micrograph of PrP amyloid fibrils. (Scale bar: 100 nm.) The width of the fibrils (arrows) was determined to be 12 ± 1 nm. (C) Model for the fold of the PrP amyloid core (residues 159–219) consistent with the present data. The native disulfide bond is shown in green, charged residues are colored red (negative) and blue (positive), and potential N-linked glycosylation sites are labeled. The only charged residue positioned in the dry interface, E211, is in hydrogen-bonding distance of Q186. The exact identity of β-strands is unknown, and orientation of the most N-terminal strands is arbitrary. (D) In-register stacking motif of nearly planar PrP monomers in the amyloid modeled with tight interdigitation of the side chains. (E) Rotated structure showing the network of intermolecular hydrogen bonding. The arrow indicates the long axis of the fibril.
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