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. 2002 Jun 17;21(12):2903-11.
doi: 10.1093/emboj/cdf303.

The yeast prion Ure2p retains its native alpha-helical conformation upon assembly into protein fibrils in vitro

Luc Bousset  1 , Neil H Thomson, Sheena E Radford, Ronald Melki
Affiliations

The yeast prion Ure2p retains its native alpha-helical conformation upon assembly into protein fibrils in vitro

Luc Bousset et al. EMBO J. .

Abstract

The yeast inheritable phenotype [URE3] is thought to result from conformational changes in the normally soluble and highly helical protein Ure2p. In vitro, the protein spontaneously forms long, straight, insoluble protein fibrils at neutral pH. Here we show that fibrils of intact Ure2p assembled in vitro do not possess the cross beta-structure of amyloid, but instead are formed by the polymerization of native-like helical subunits that retain the ability to bind substrate analogues. We further show that dissociation of the normally dimeric protein to its constituent monomers is a prerequisite for assembly into fibrils. By analysing the nature of early assembly intermediates, as well as fully assembled Ure2p fibrils using atomic force microscopy, and combining the results with experiments that probe the fidelity of the native fold in protein fibrils, we present a model for fibril formation, based on assembly of native-like monomers, driven by interactions between the N-terminal glutamine and asparagine-rich region and the C-terminal functional domain. The results provide a rationale for the effect of mutagenesis on prion formation and new insights into the mechanism by which this, and possibly other inheritable factors, can be propagated.

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Figures

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Fig. 1. X-ray structure of the C-terminal domain (residues 95–354) of Ure2p. The site of Cys221 engineered into the protein to stabilize the dimer is shown in yellow. The two subunits in the dimer are coloured blue and green. The structure also shows the position of GSH (in red), which binds between two subdomains within each subunit. The figure was drawn with Molscript (Kraulis, 1991) and Raster3d using the crystal coordinates deposited in the Protein Data Bank, accession No. 1jzr (Bousset et al., 2001b).
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Fig. 2. Ure2p loses its ability to assemble into fibrils when cross-linked in a dimeric form. (A) Analysis on a non-reducing 10% SDS– polyacrylamide gel of Ure2pC221 in the presence of the reducing agent DTT (lane 1) and following its cross-linking by H2O2 (lane 2). Cross-linked Ure2pC221 following its reduction in the presence of β-mercaptoethanol (lane 3). The molecular weight markers are shown on the left. The apparent molecular mass of cross-linked Ure2pC221 (116 kDa) is higher than the expected molecular mass of Ure2p dimer (80.4 kDa). This must reflect an abnormal shape of the cross-linked dimer. The polypeptide with apparent molecular mass 70 kDa is a contaminant. (B and C) Negative-stained electron micrographs of oligomers made from the reduced (B) and cross-linked (C) forms of Ure2pC221. Bar, 0.2 µm.
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Fig. 3. Soluble and fibrillar Ure2p have similar secondary structural contents. FTIR spectra of soluble full-length Ure2p (A), Ure2p 95–354 (B) and fibrils assembled from full-length Ure2p (C). Curve fit spectra are presented in each case.
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Fig. 4. Fibrillar Ure2p binds GSH in a manner similar to native soluble Ure2p. (A) Saturation of Ure2p fibrils by ADAN–GSH (solid symbols). The solid curve depicts the calculated isotherm for binding of ADAN–GSH to a single category of sites (10 µM) in Ure2p fibrils and an equilibrium dissociation constant of 25 µM. Inset: double-reciprocal plot of the data. Open symbols and dashed line: titration of fibrils with ADAN–GSH in the presence of 2 M GdnHCl. Under these conditions, the fibrils remain assembled, yet lose the ability to bind the ligand. (B) Competition between GSH and ADAN–GSH for binding to fibrils of Ure2p. ADAN–GSH (20 µM) bound to fibrils of Ure2p (3.8 µM) was chased by increasing concentrations of unlabelled GSH in 50 mM Tris pH 7.5 and 100 mM KCl.
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Fig. 5. AFM image analysis of the formation of Ure2p fibrils. (A) Time course of Ure2p (60 µM) assembly into fibrils in 50 mM Tris pH 7.5 and 100 mM KCl monitored by thioflavin-T binding. (B) Tapping-mode AFM amplitude image (error signal) of Ure2p oligomers at an early stage of assembly, shown by the open arrow in (A). (C) Tapping-mode AFM amplitude image (error signal) of Ure2p fibrils at a late stage of assembly, shown by the closed arrow in (A). (D) Tapping-mode AFM height image of the image shown in (C). (E) Fibril height profile along the axis of the fibril in the white frame in (D) showing its periodicity. The repeating unit of the fibril is 30 nm. (F) Cross-section profile for the fibrils presented in (D) performed in the region indicated by the dotted line. The height of the two fibrils is 11 nm and their diameter at half-height is 38 nm. The images were acquired in air. Scale bar, 200 nm.
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Fig. 6. Tapping-mode AFM image analysis using height data (z-piezo movement) of native, GdnHCl- and proteinase K-treated Ure2p fibrils. (A, E and I) AFM height images of native, GdnHCl- (4 M) and proteinase K- (10 µg/ml) treated Ure2p fibrils (60 µM), respectively. The dimensions of the fibrils were analysed as described in Materials and methods. The panels show the height, width and periodicity distributions, respectively, for native fibrils (B, C and D), fibrils treated with 4 M GdnHCl (F, G and H) and fibrils treated with proteinase K (J, K and L). The number of individual objects measured is 3745 in (B), 3435 in (C), 158 in (D), 444 in (F), 407 in (G), 29 in (H), 356 in (J), 95 in (K) and 23 in (L).
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Fig. 7. Molecular models for the assembly of Ure2p into fibrils. The proposed models are based on the observations presented here that: (i) dissociation of the Ure2p dimer occurs prior to assembly into fibrils; (ii) the fibrils formed under the conditions used lack a cross β-core; and (iii) the observation that Ure2p retains a native C-terminal domain in the fibrils. The dissociation of native Ure2p dimer exposes a hydrophobic surface area to the solvent (red). This area can establish a large number of hydrogen bonds with the flexible N-terminal domain (blue) from either the same Ure2p molecule or another Ure2p polypeptide chain, leading to intra- (case A) or inter- (case B) molecular interactions, respectively. In case A, the N-terminal domain binds to the same monomer, forming a surface that favours its interaction with the flexible cap region (green) from another Ure2p molecule, leading to the association of two Ure2p monomers through non-native interactions. In case B, the hydrophobic surface area exposed by dissociation of a given Ure2p monomer interacts with the N-terminal domain from another Ure2p polypeptide chain, forming a non-native dimer. In both cases, assembly can proceed indefinitely by addition of Ure2p monomers to both ends of the non-native dimer, given that each additional monomer exposes a binding site similar to that masked by its association. The oligomers generated are helical since two neighbouring monomers have the same relative orientation. In both cases, the GSH binding site (pink) is maintained in a native conformation in the fibril and is available for binding its ligand.
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