NMR-detected hydrogen exchange and molecular dynamics simulations provide structural insight into fibril formation of prion protein fragment 106-126

Abstract

PrP106-126, a peptide corresponding to residues 107-127 of the human prion protein, induces neuronal cell death by apoptosis and causes proliferation and hypertrophy of glia, reproducing the main neuropathological features of prion-related transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Although PrP106-126 has been shown to form amyloid-like fibrils in vitro, their structural properties have not been elucidated. Here, we investigate the conformational characteristics of a fibril-forming fragment of the mouse prion protein, MoPrP106-126, by using electron microscopy, CD spectroscopy, NMR-detected hydrogen-deuterium exchange measurements, and molecular dynamics simulations. The fibrils contain approximately 50% beta-sheet structure, and strong amide exchange protection is limited to the central portion of the peptide spanning the palindromic sequence VAGAAAAGAV. Molecular dynamics simulations indicate that MoPrP106-126 in water assumes a stable structure consisting of two four-stranded parallel beta-sheets that are tightly packed against each other by methyl-methyl interactions. Fibril formation involving polyalanine stacking is consistent with the experimental observations.

Fig. 1.

Electron micrograph of a negatively stained preparation of the peptide MoPrP106–126 in 100 mM sodium acetate/150 mM NaCl, pH 5.5/50% (vol/vol) acetonitrile. The average length and width of the fibrils is 0.2 μm and 80 Å, respectively.

Fig. 2.

CD spectra of MoPrP106–126 in 100 mM sodium acetate/150 mM NaCl, pH 5.5/50% (vol/vol) acetonitrile, recorded at 25°C in the absence (•) and presence (○) of 75% (vol/vol) trifluoroethanol. MRW, mean residue weight.

Fig. 3.

NH–C^αH region of a TOCSY spectrum of MoPrP106–126 in 95% DMSO-d₆/4.5% D₂O/0.5% dichloroacetic-d₂ acid (vol/vol/vol), pH 5.0, uncorrected.

Fig. 4.

H/D exchange protection factors of individual amide protons in MoPrP106–126 amyloid fibrils. Bars indicate the protection factors, and ○ indicate H/D exchange time constants. Reliable exchange rates could not be measured for the Gly-113 and Gly-123 NH peaks because of their proximity to the water resonance.

Fig. 5.

Representative structure of the octameric complex of PrP106–126 calculated by using MD simulations. All peptides are oriented with the N terminus at the bottom. (a) Front view, using functional coloring for the top layer (strands A–D) and gray for the bottom layer (strands E–H). (b) Side view, using the same coloring scheme as in a. (c) Side view, emphasizing the methyl packing interactions in the core of the complex. Residues (His-110 to Val-120) on two opposite strands (B and F) are shown as a ball-and-stick representation (including H), and other residues are shown as a wire diagram. (d) Schematic ribbon diagram of the predicted PrP106–126 fibril structure.

Fig. 6.

Distribution of H bonds (<2.2 Å) that were formed during the simulation of octameric PrP106–126 in the palindromic (•) and nonpalindromic (○) regions of the sequence. Eight 21-residue peptides (A–H; see Fig. 5d) are aligned along the horizontal and vertical axes.