Assembly of the yeast prion Ure2p into protein fibrils. Thermodynamic and kinetic characterization

The [URE3] phenotype in Saccharomyces cerevisiae propagates by a prion mechanism, involving the aggregation of the normally soluble and highly helical protein Ure2. Previous data have shown that the protein spontaneously forms in vitro long, straight, insoluble fibrils at neutral pH that are similar to amyloids in that they bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis. These fibrils are not amyloids as they are devoid of a cross-beta core. Here we further document the mechanism of assembly of Ure2p into fibrils. The critical concentration for Ure2p assembly is measured, and the minimal size of the nuclei that are the precursors of Ure2p fibrils is determined. Our data indicate that the assembly process is irreversible. As a consequence, the critical concentration is very low. By analyzing the elongation rates of preformed fibrils and combining the results with single-fiber imaging experiments of a variant Ure2p labeled by fluorescent dyes, we reveal the polarity of the fibrils and differences in the elongation rates at their ends. These results bring novel insight in the process of Ure2p assembly into fibrils and the mechanism of propagation of yeast prions.     

Structure of the prion Ure2p in protein fibrils assembled in vitro

The Ure2 protein from the yeast Saccharomyces cerevisiae has prion properties. In vitro and at neutral pH, soluble Ure2p spontaneously forms long, straight, insoluble protein fibrils. Two models have been proposed to account for the assembly of Ure2p into protein fibrils. The "amyloid backbone" model postulates that a segment ranging from 40 to 70 amino acids in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second model hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and/or intramolecular interactions between Ure2p monomers. Here, we performed a cysteine scan on residues located in the N- and C-terminal parts of Ure2p to determine whether these domains interact. Amino acid sequences centered around residue 6 in the N-terminal domain of Ure2p and residue 137 in the C-terminal moiety interacted at least transiently via intramolecular interactions. We documented the assembly properties of a Ure2p variant in which a disulfide bond was established between the N- and C-terminal domains and showed that it possesses assembly properties indistinguishable from those of wild-type Ure2p. We probed the structure of Ure2pC6C137 within the fibrils and demonstrate that the polypeptide is in a conformation similar to that of its soluble assembly-competent state. Our results constitute the first structural characterization of the N-terminal domain of Ure2p in both its soluble assembly-competent and fibrillar forms. Our data indicate that the flexibility of the N-terminal domain and conformational changes within this domain are essential for fibril formation and provide new insight into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] phenotype in yeast.     

Hydrogen/deuterium exchange mass spectrometric analysis of conformational changes accompanying the assembly of the yeast prion Ure2p into protein fibrils

The Ure2 protein from baker's yeast (Saccharomyces cerevisiae) has prion properties. In vitro, at neutral pH, soluble Ure2p forms long, twisted fibrils. Two models have been proposed to account for Ure2p polymerization. The first postulates that a segment of 70 amino acid residues in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and intramolecular interactions. The knowledge of the three-dimensional structure of the fibrillar form of Ure2p is critical for understanding the molecular events leading to the polymerization of soluble Ure2p into fibrils and hence for the design of inhibitors that might have therapeutic potential as yeast prions possessing domains rich in N and Q residues, similar to huntingtin. Solvent-accessibility studies using hydrogen/deuterium exchange monitored by mass spectrometry (HXMS) can provide insights into the structure of the fibrillar form of Ure2p and characterize at the molecular level the conformational rearrangements that occur upon assembly, in particular through the identification of protected regions and their localization in the overall structure of the protein. We have analyzed the changes in Ure2p structure associated with its assembly into fibrils using HXMS. The deuterium incorporation profile along the sequence allows the identification of the regions that exhibit the most important conformational change. Our data reveal that Ure2p undergoes minor structural changes upon assembly. While polypeptides [82-92] and [13-37] exhibit significant increased and decreased exposure to the solvent, respectively, no marked change was observed for the rest of the protein upon assembly. Our results afford new insights into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] element in yeast.     

Structure and assembly properties of the yeast prion Ure2p

The non-Mendelian phenotype [URE3] is due to a transmissible conformational change of the protein Ure2. The infectious protein form of Ure2p has lost its function and gained the capacity to transform the active form of the protein into an inactive form. The molecular basis of this conversion process is unknown. There are however indications that the conformational changes at the origin of the propagation of the inactive form of Ure2p in yeast cells are similar to those at the origin of the transition of PrPC into the scrapie-associated PrPSc form of the protein. To better understand the nature of the conformational changes at the origin of prion propagation, we have purified, characterized biochemically, examined the assembly properties and solved the crystal structure of Ure2p. Our data are presented below and a number of conclusions dealing with the molecular basis of the conversion of soluble Ure2p into its amyloid-forming state are derived.     

Folding of prion protein to its native alpha-helical conformation is under kinetic control

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric alpha-helical or oligomeric beta-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the beta-rich isoform of MoPrP is thermodynamically more stable than the native alpha-helical isoform. The conformational transition from the alpha-helical to beta-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the alpha-helical isoform and folds directly to the thermodynamically more stable beta-rich isoform. Our data demonstrate that the folding of the prion protein to its native alpha-helical monomeric conformation is under kinetic control.     

Molecular chaperones and the assembly of the prion Ure2p in vitro

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94-354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].     

Insights into the architecture of the Ure2p yeast protein assemblies from helical twisted fibrils

The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.     

Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.     

The native-like conformation of Ure2p in fibrils assembled under physiologically relevant conditions switches to an amyloid-like conformation upon heat-treatment of the fibrils

The [URE3] phenotype in the yeast Saccharomyces cerevisiae is inherited by a prion mechanism involving self-propagating Ure2p aggregates. It is believed that assembly of intact Ure2p into fibrillar polymers that bind Congo Red and show yellow-green birefringence upon staining and are resistant to proteolysis is the consequence of a major change in the conformation of the protein. We recently dissected the assembly process of Ure2p and showed the protein to retain its native alpha-helical structure upon assembly into protein fibrils that are similar to amyloids in that they are straight, bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis (). Here we further show using specific ligand binding, FTIR spectroscopy and X-ray fiber diffraction that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta core. The X-ray fiber diffraction pattern of these fibrils reveals their well-defined axial supramolecular order. By analyzing the effect of heat-treatment on Ure2p fibrils we bring evidences for a large conformational change that occurs within the fibrils with the loss of the ligand binding capacity, decrease of the alpha helicity, the formation of a cross-beta core and the disappearance of the axial supramolecular order. The extent of the conformational change suggests that it is not limited to the N-terminal part of Ure2p polypeptide chain. We show that the heat-treated fibrils that possess a cross-beta core are unable to propagate their structural characteristic while native-like fibrils are. Finally, the potential evolution of native-like fibrils into amyloid fibrils is discussed.     

The yeast prion protein Ure2: insights into the mechanism of amyloid formation

Ure2, a regulator of nitrogen metabolism, is the protein determinant of the [URE3] prion state in Saccharomyces cerevisiae. Upon conversion into the prion form, Ure2 undergoes a heritable conformational change to an amyloid-like aggregated state and loses its regulatory function. A number of molecular chaperones have been found to affect the prion properties of Ure2. The studies carried out in our laboratory have been aimed at elucidating the structure of Ure2 fibrils, the mechanism of amyloid formation and the effect of chaperones on the fibril formation of Ure2.     

The yeast prion protein Ure2 shows glutathione peroxidase activity in both native and fibrillar forms

Ure2p is the precursor protein of the Saccharomyces cerevisiae prion [URE3]. Ure2p shows homology to glutathione transferases but lacks typical glutathione transferase activity. A recent study found that deletion of the Ure2 gene causes increased sensitivity to heavy metal ions and oxidants, whereas prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochemical characterization of the purified protein. Here we use steady-state kinetic methods to characterize the multisubstrate peroxidase activity of Ure2p using GSH with cumene hydroperoxide, hydrogen peroxide, or tert-butyl hydroperoxide as substrates. Glutathione-dependent peroxidase activity was proportional to the Ure2p concentration and showed optima at pH 8 and 40 degrees C. Michaelis-Menten behavior with convergent straight lines in double reciprocal plots was observed. This excludes a ping-pong mechanism and implies either a rapid-equilibrium random or a steady-state ordered sequential mechanism for Ure2p, consistent with its classification as a glutathione transferase. The mutant 90Ure2, which lacks the unstructured N-terminal prion domain, showed kinetic parameters identical to wild type. Fibrillar aggregates showed the same level of activity as native protein. Demonstration of peroxidase activity for Ure2 represents important progress in elucidation of its role in vivo. Further, establishment of an in vitro activity assay provides a valuable tool for the study of structure-function relationships of the Ure2 protein as both a prion and an enzyme.     

Pressure denaturation of the yeast prion protein Ure2

Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure. Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl. Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state. Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar.     

Parallel beta-sheets and polar zippers in amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p

We report the results of solid-state nuclear magnetic resonance (NMR) and atomic force microscopy measurements on amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p (Ure2p(10)(-)(39)). Measurements of intermolecular (13)C-(13)C nuclear magnetic dipole-dipole couplings indicate that Ure2p(10)(-)(39) fibrils contain in-register parallel beta-sheets. Measurements of intermolecular (15)N-(13)C dipole-dipole couplings, using a new solid-state NMR technique called DSQ-REDOR, are consistent with hydrogen bonds between side chain amide groups of Gln18 residues. Such side chain hydrogen bonding interactions have been called "polar zippers" by M. F. Perutz and have been proposed to stabilize amyloid fibrils formed by peptides with glutamine- and asparagine-rich sequences, such as Ure2p(10)(-)(39). We propose that polar zipper interactions account for the in-register parallel beta-sheet structure in Ure2p(10)(-)(39) fibrils and that similar peptides will also exhibit parallel beta-sheet structures in amyloid fibrils. We present molecular models for Ure2p(10)(-)(39) fibrils that are consistent with available experimental data. Finally, we show that solid-state (13)C NMR chemical shifts for (13)C-labeled Ure2p(10)(-)(39) fibrils are insensitive to hydration level, indicating that the fibril structure is not affected by the presence or absence of bulk water.     

Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Chem. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.     

Characterization of beta-sheet structure in Ure2p1-89 yeast prion fibrils by solid-state nuclear magnetic resonance

Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole-dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N-13C and 13C-13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole-dipole couplings indicate that the beta-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel beta-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1-89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218-289 of the HET-s prion protein of Podospora anserina [as originally reported in Siemer, A. B., Ritter, C., Ernst, M., Riek, R., and Meier, B. H. (2005) Angew. Chem., Int. Ed. 44, 2441-2444 and confirmed by measurements reported here] by factors of three or more, indicating a lower degree of structural order at the molecular level in Ure2p1-89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biological function of HET-s in heterokaryon incompatibility. Analysis of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid beta-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.     

The yeast prion protein Ure2: structure, function and folding

The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metabolism and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymatic function appears to be maintained. A number of factors are found to affect the prion properties of Ure2, including mutation and expression levels of molecular chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.     

Hierarchical organization in the amyloid core of yeast prion protein Ure2

Formation of amyloid fibrils is involved in a range of fatal human disorders including Alzheimer, Parkinson, and prion diseases. Yeast prions, despite differences in sequence from their mammalian counterparts, share similar features with mammalian prions including infectivity, prion strain phenomenon, and species barrier and thus are good model systems for human prion diseases. Yeast prions normally have long prion domains that presumably form multiple β strands in the fibril, and structural knowledge about the yeast prion fibrils has been limited. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the structures of amyloid fibrils of Ure2 prion domain. We show that 15 spin-labeled Ure2 mutants, with spin labels at every 5th residue from position 5 to position 75, show a single-line or nearly single-line feature in their EPR spectra as a result of strong spin exchange interactions. These results suggest that a parallel in-register β structure exists at these spin-labeled positions. More interestingly, we also show that residues in the segment 30-65 have stronger spin exchange interactions, higher local stability, and lower solvent accessibility than segments 5-25 and 70-75, suggesting different local environment at these segments. We propose a hierarchical organization in the amyloid core of Ure2, with the segment 30-65 forming an inner core and the segments 5-25 and 70-75 forming an outer core. The hierarchical organization in the amyloid core may be a structural origin for polymorphism in fibrils and prion strains.     

The N-terminal prion domain of Ure2p converts from an unfolded to a thermally resistant conformation upon filament formation

According to the "amyloid backbone" model of Ure2p prionogenesis, the N-terminal domain of Ure2p polymerizes to form an amyloid filament backbone surrounded by the C-terminal domains. The latter domains retain their native glutathione-S-transferase (GST)-like fold but are sterically inactivated from their regulatory role in nitrogen catabolism. We have tested this model by differential scanning calorimetry of soluble and filamentous Ure2p and of soluble C-terminal domains, combined with electron microscopy. As predicted, the C-terminal domains respond to thermal perturbation identically in all three states, exhibiting a single endotherm at 76 degrees C. In contrast, no thermal signal was associated with the N-terminal domains: in the soluble state of Ure2p, because they are unfolded; in the filamentous state, because their robust amyloid conformation resists heating to 100 degrees C.     

Small angle X-ray scattering study of the yeast prion Ure2p

The GdmCl-induced equilibrium unfolding and dissociation of the dimeric yeast prion protein Ure2, and its prion domain deletion mutants Delta 15-42Ure2 and 90Ure2, was studied by small angle X-ray scattering (SAXS) using synchrotron radiation and by chemical cross-linking with dithiobis(succinimidyl propionate) (DTSP). The native state is globular and predominantly dimeric prior to the onset of unfolding. R(g) values of 32 and 45A were obtained for the native and 5M GdmCl denatured states of Delta 15-42Ure2, respectively; the corresponding values for 90Ure2 were 2-3A lower. SAXS suggests residual structure in the 4M GdmCl denatured state and chemical cross-linking detects persistence of dimeric structure under these conditions. Hexamers consisting of globular subunits could be detected by SAXS at high protein concentration under partially denaturing conditions. The increased tendency of partially folded states to form small oligomers points to a mechanism for prion formation.     

Packing of the prion Ure2p in protein fibrils probed by fluorescence X-ray near-edge structure spectroscopy at sulfur K-edge

The soluble protein Ure2p from the yeast Saccharomyces cerevisiae assembles in vitro into straight and insoluble protein fibrils, through subtle changes of conformation. Whereas the structure of soluble Ure2p has been revealed by X-ray crystallography, further characterization of the structure of insoluble Ure2p fibrils is needed. We performed X-ray absorption near-edge spectroscopy (XANES) at the sulfur K-edge to probe the state of Cys221 in the fibrillar form of Ure2pC221 and provide structural information on the structure of Ure2p within fibrils. Although the Ure2p dimer dissociation into its constituent monomers has proven to be a prerequisite for assembly into fibrils, we showed the ability of every Ure2pC221 monomer to establish disulfide bonds upon incubation of the fibrils under oxidizing conditions. Our result indicates either that the constituent unit of the fibrillar form of the protein is a dimeric Ure2p or that the fibrils are made of protofilaments assembled in such a way that the residue C221 from a Ure2p molecule in one protofilament is located in the vicinity of a C221 residue from another molecule belonging to a neighbor protofilament.