the native-like conformation of ure2p in fibrils assembled under physiologically relevant conditions...

The native-like conformation of Ure2p in fibrils assembledunder physiologically relevant conditions switches to an

amyloid-like conformation upon heat-treatment of the fibrils

Luc Bousset,a Fatma Briki,b Jean Doucet,b,c and Ronald Melkia,*

a Laboratoire d�Enzymologie et Biochimie Structurales, CNRS, Baatiment 34 Avenue de la Terrasse, Gif-sur-Yvette Cedex FR-91198, Franceb Laboratoire d�Utilisation du Rayonnement Electromagn�eetique, Universit�ee Paris Sud, BP 34, Orsay 91898, France

c European Synchrotron Radiation Facility, Boııte Postale 220, Grenoble Cedex 38043, France

Received 11 September 2002, and in revised form 6 November 2002

Abstract

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 bire-

fringence 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 a-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 (Bousset et al., 2002). 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-b core. The X-ray

fiber diffraction pattern of these fibrils reveals their well-defined axial supramolecular order. By analyzing the effect of heat-treat-

ment 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 a helicity, the formation of a cross-b 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-b 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.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Prion; Saccharomyces cerevisiae; Ure2p; Assembly; Amyloid fibrils; Conformational transition; X-ray fiber diffraction

1. Introduction

An intriguing group of neurodegenerative diseases,

among which transmissible spongiform encephalopa-

thies (TSEs) in mammals are believed to be due to the

infectious protein (prion) PrP (Bolton et al., 1982). In

the yeast Saccharomyces cerevisiae, at least four proteinscause heritable, self-perpetuating, changes in phenotype.

These proteins are Ure2p, Sup35p, and Rnq1p, they are

responsible of the phenotypes [URE3] (Lacroute, 1971),

½PSIþ� (Cox, 1965), and ½PINþ� (induction of PSIþ)

(Derkatch et al., 2001; Sondheimer and Lindquist,

2000), respectively. A fourth protein, New1p (Santoso

et al., 2000), is believed to have prion properties but no

specific phenotype.

Ure2p is involved in a signal transduction pathway

that regulates nitrogen catabolism (Magasanik, 1992).

[URE3] is due to the self-propagation of altered formsof Ure2p (Wickner, 1994). In wild type cells, Ure2p is

dispersed throughout the cytoplasm while it forms large

globular or filiform aggregates in cells carrying the

[URE3] phenotype (Edskes et al., 1999; Fernandez-

Bellot et al., 2000). Overexpression of Ure2p 65 N-ter-

minal amino acid residues in wild type cells is sufficient

to induce de novo appearance of [URE3] while the ex-

pression of the complementary C-terminal region in

Journal of Structural Biology 141 (2003) 132–142

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* Corresponding author. Fax: +33-1-69-82-31-29.

E-mail addresses: [email protected] (J. Doucet), melki@-

lebs.cnrs-gif.fr (R. Melki).

1047-8477/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S1047-8477(02)00606-8

mail to: [email protected]

yeast cells lacking the URE2 gene restores the functionof the gene (Masison and Wickner, 1995). This is at the

origin of the definition of a prion-forming (residues 1–

65) and a functional (residues 65–354) part. The physical

boundary between the two domains was redefined and

their respective properties accessed using purified solu-

ble native Ure2p (Thual et al., 1999). The N-terminal

region extending from residues 1–93 is poorly structured

while the C-terminal region extending from residues 94–354 is compactly folded (Thual et al., 1999). The crystal

structure of Ure2p 95–354 was solved (Bousset et al.,

2001a; Umland et al., 2001). Ure2p 95–354 is highly a-helical. It is formed by two sub-domains and has a fold

similar to that of glutathione-S-transferases (GSTs)

(Board et al., 2000). It binds glutathione (GSH) and

related compounds with high affinity in a cleft running

along the domain interface that resembles the active siteof GSTs (Bousset et al., 2001b).

We have shown previously that authentic Ure2p can

assemble in vitro into fibrils under physiologically rele-

vant conditions (Thual et al., 2001, 1999). Assembly is

accompanied by an increase in the resistance to prote-

olysis (Thual et al., 1999). Interestingly however, the

degradation patterns of soluble and assembled Ure2p

are very similar suggesting that the protein remains in anative-like conformation in the fibrils. Ure2p fibrils bind

the dye Congo red (Thual et al., 1999). This finding by

itself does not mean that the fibrils are amyloids since

binding of Congo red is not specific of amyloids

(Khurana et al., 2001). Binding is however accompanied

by the amyloid characteristic yellow-green birefringence

in cross-polarized light (Taylor et al., 1999).

A large number of proteins that possess unrelatedstructures form amyloid-like fibrils under given experi-

mental conditions (Carrell and Gooptu, 1998; Fandrich

et al., 2001; Krebs et al., 2000; Lim et al., 2000;

Morozova-Roche et al., 2000). This suggests that a

common factor drive the formation of fibrils despite

their highly differing primary structures. Up to now, this

factor was thought to be a partial unfolding–refolding

process (Dobson, 1999), leading to the formation ofintermolecular bonding through b-sheets. These b-sheetsform a cross-b core running all along the fibril with the

hydrogen-bonded polypeptide chains running normal to

the fibril direction and show a characteristic cross-bmeridional 4.7–4.8�AA reflection in X-ray fiber diffraction

patterns (Sunde et al., 1997).

We recently dissected the assembly process of Ure2p

and showed the protein to retain its native a-helicalstructure upon assembly into protein fibrils (Bousset

et al., 2002). The absence of a major conformational

change upon Ure2p assembly into fibrils that exhibit

several characteristics of amyloids can be readily ex-

plained if one assumes that the conformational change

that drives Ure2p assembly only affects the poorly

structured N-terminal domain of the protein. This

region extends over 65–94 amino acid residues out of thetotal 354 amino acids of Ure2p.

Here we further show using X-ray fiber diffraction

that Ure2p fibrils assembled under physiologically rele-

vant conditions are devoid of a cross-b structure. While

trying to improve Ure2p fibrils alignment to elucidate

the structure of the fibrillar form of Ure2p, we observed

a change in their architectures. While the a-helicalcontent of Ure2p in fibrils assembled at 20 �C was verysimilar to that of the native protein, fibrils exposed

to 60 �C were essentially made of b-sheets. The two

kinds of fibrils are indistinguishable in the electron

microscope. The observation of protein fibrils with dif-

ferent structures and morphologies, either within the

same preparation, or under different physico-chemical

conditions, has already been reported (Kad et al., 2001),

but the absence of a cross-b core signal is withoutprecedent for amyloid fibrils. The absence of a cross

b-sheet core in Ure2p fibrils obtained under physiolog-

ical conditions raises the question of the necessity of

such a core in fibril formation and of its role in their

stabilization.

Upon heating the native-like fibrils in solution, a

change in their architecture occurs with the formation of

a cross b-sheet core. The proteinase K degradationpatterns of Ure2p in native-like and heated fibrils are

unrelated. Moreover, while Ure2p in native-like fibrils

binds GSH in a manner indistinguishable from that of

the native unassembled form of the protein (Bousset et

al., 2001a), the heat-treated fibrils have lost their binding

capacity. All together, our data indicate that a major

change in the conformation of Ure2p within the fibrils

occurs upon heating. This change corresponds to thetransition from a mainly a-helical to a mainly b-sheetstructure, i.e., a transition from a helical polymer to an

amyloid fibril. Interestingly, while native-like fibrils

propagate in a catalytic manner upon dilution in the

presence of soluble Ure2p, heat-treated fibrils are inert

end products. This indicates that the cross-b rich Ure2p

fibrils are irrelevant to the prion concept while native-

like fibrils are.

2. Materials and methods

2.1. Purification and crystallization

Full length Ure2p and its C-terminal region extending

from residues 95–354 were overexpressed in Escherichiacoli and purified as previously described (Thual et al.,

2001, 1999). Protein concentrations were determined

spectrophotometrically (HP 8453 diode array spectro-

photometer, Hewlett-Packard) using an extinction co-

efficient of 0.67mg cm2 at 280 nm and a molecular

weight of 40 200 or by the Bradford methods (Bradford,

1976). Crystals of full length Ure2p and Ure2p 95–354

L. Bousset et al. / Journal of Structural Biology 141 (2003) 132–142 133

were obtained using the crystallization conditions de-scribed in (Bousset et al., 2001a).

2.2. Assembly of Ure2p into fibrils

Assembly of Ure2p into fibrils was monitored using a

thioflavin-T binding assay (McParland et al., 2000).

Ure2p (60 lM) in 20mM Tris, pH 7.5, 100mM KCl was

incubated at 7 �C. At regular time intervals, 8 ll aliquotswere removed from the solution, mixed with 300 ll of

thioflavin-T (10 lM) from Sigma and incubated at 20 �Cfor 10min. Thioflavin-T binding was measured by av-

eraging the emission signal over 30 s using an AM-

INCO-Bowman series2 spectrofluorometer set at 440 nm

(excitation) and 480 nm (emission). Ure2p fibrils were

negatively stained on carbon-coated grids (200 mesh)

with 1% uranyl acetate and examined in a Philips EM410 electron microscope.

2.3. Binding of acetyl-2-dimethylaminonaphtalene gluta-

thione to Ure2p fibrils

Glutathione (Roche), 50mM, in HEPES, pH 7.5,

was incubated with 20mM 6-bromoacetyl-2-dim-

ethylaminonaphtalene (Molecular Probes, Eugene, OR)for 2 h at room temperature in order to allow the for-

mation of a thioether bond between the SH group of the

molecule and the probe. Binding of acetyl-2-dim-

ethylaminonaphtalene-glutathione (ADAN-glutathione)

to untreated or heat-treated Ure2p fibrils in 20mM Tris,

pH 7.5, 100mM KCl was monitored in a 10� 2mm

quartz cuvette (Hellma) thermostated at 20 �C in a

AMINCO-Bowman series2 spectrofluorometer. Theexcitation and emission monochromators were set at 380

and 500 nm, respectively.

2.4. Proteolytic digestions

Assembly of Ure2p (2.0mg/ml) in 20mM Tris, pH

7.5, 100mM KCl into fibrils was allowed to completion

by incubating soluble Ure2p for 80 h at 7 �C. The samesample was then split into two aliquots. The first was

incubated without any additional treatment at 37 �Cwith Proteinase K from Roche (2.4 lg/ml), while the

second was incubated for 60min at 60 �C prior to the

proteolytic treatment. Aliquots from each sample were

removed at different time intervals following addition of

the protease and transferred into Eppendorf tubes

maintained at 95 �C containing sample denaturing buffer(50mM Tris–HCl, pH 6.8, 4% SDS, 2% b-mercap-

toethanol, 12% glycerol 0.01% bromophenol blue) in

order to arrest immediately the cleavage reaction. After

incubation of each tube for 10min at 95 �C, the samples

were processed to monitor the time course of Ure2p

cleavage by SDS–PAGE (Laemmli, 1970).

2.5. X-ray diffraction (XRD)

Assembly of Ure2p (4.0mg/ml) in 20mM Tris, pH

7.5, 100mM KCl into fibrils was allowed to completion

by incubating soluble Ure2p for 48 h at 20 �C. The

sample was then dialyzed against water for 4 h and split

into two aliquots. One of the aliquots was heated for 1 h

at 60 �C the other was untreated. About 25 ll of each

aliquot were introduced into glass capillaries 1.5mmdiameter. The fibrils were aligned either in a magnetic

field (1 T) at 20 �C for one week or evaporated at room

temperature. The material was concentrated following

these treatments into a thin disk of about 0.3mm

thickness, delimited by the capillary cross-section.

X-ray data collection was carried out on two syn-

chrotron beamlines, D43 at LURE (Orsay, France) and

ID14-EH1 at the European Synchrotron RadiationFacility (Grenoble, France). D43 beamline uses a colli-

mated and monochromatic incident beam (0.5mm di-

ameter cross-section, wavelength 1.45�AA) selected by a

Ge (1 1 1) bent monochromator. A transmission geom-

etry setting was chosen with a sample-detector distance

of 100mm, giving access to periodicities 30 and 2.5�AA.

The two-dimensional scattering patterns were recorded

on FUJI PhosphorImage plates located perpendicular tothe incident beam and read on a AMERSHAM scanner

(STORM) with a 200 lm pixel size and 0.08� angular

resolution. The X-ray beam (wavelength 0.934�AA) had a

diameter of 50 lm on ID14-EH1 and allowed the

analysis of sample sections 100 times smaller than at

LURE, thus increasing the probability to focus

the beam on well-oriented regions. The combination of

the 80 lm pixel size of the MarResearch CCD detectorwith the 150mm sample-to-detector distance gave access

to periodicities between 80 and 2�AA, with an angu-

lar resolution of 0.02�. Data processing involved sub-

traction of the scattering pattern due to the glass

capillary.

2.6. Fourier transform infrared spectrometry (FT-IR)

Infrared spectra were collected using a Spectra Tech

infrared microscope equipped with a small aperture

ð5� 5lm2Þ at the synchrotron radiation facility,

Beamline MIRAGE (Polack et al., 1999) at LURE,

Orsay, France. Spectra of untreated and heat-treated

Ure2p fibrils recovered by sedimentation at 100 000g for

15min and that of crystals of Ure2p 95–354 were re-

corded at 8 cm�1 resolution. Five hundred and twelvespectra were collected before performing Fourier

transform. A linear baseline subtraction was performed

to account for the gradual decay in the beam intensity

due to synchrotron source decay. The amide I band

(1600–1700 cm�1) of the spectra was subjected to a fit-

ting procedure using six gaussian distributions centered

134 L. Bousset et al. / Journal of Structural Biology 141 (2003) 132–142

at the frequencies of well characterized secondarystructures (Stuart, 1997). Each secondary structure is

characterized by its frequency and by an interval of a

given width. During the fitting procedure the peak po-

sitions were free to vary inside that interval. Each peak

width was limited to 25 cm�1 while peak height was free.

The fitting was achieved using the fitting �module� of theOrigin package. It is worthwhile to note that all the

frequencies were present on the second derivative of thespectra. The contribution of each curve to the amide I

band was assessed by integrating the area under the

curve and then normalizing to the total area under the

amide I band.

3. Results

3.1. Untreated and heat-treated Ure2p fibrils differ in their

ligand binding capacities

Native unassembled Ure2p binds with high affinity

ADAN-GSH, a substrate analogue of GSH (Bousset et

al., 2001b). The GSH binding site is located in the

functional domain of the protein, which is made of twosub-domains connected by a short linker region (Bousset

et al., 2001a), each of which is involved in substrate

binding site. ADAN-GSH is thus an excellent tool for

probing whether the fibrils contain native-like subunits.

Fig. 1. Native-like and heat-treated Ure2p fibrils differ by their binding capacity of GSH but are indistinguishable on electron micrographs. (A)

Saturation of native-like (�) and heat-treated (N) Ure2p fibrils (1mg/ml) by ADAN-GSH. (B and C) Negative stained electron micrographs of

native-like and heat-treated Ure2p fibrils, respectively. Bar, 0.2lm.


Fig. 1A shows the saturation by ADAN-GSH of Ure2pfibrils generated under physiologically relevant condi-

tions. Comparison of the data with that obtained for

soluble dimeric Ure2p (Bousset et al., 2001b) strongly

suggests that the C-terminal domain of the protein re-

tains a native conformation within the fibrils. The

ADAN-GSH binding capacity of Ure2p is lost upon

incubation of native-like fibrils in solution at 60 �C for

1 h (Fig. 1A). We conclude from this observation that aconformational rearrangement occurs during heat

treatment that reveals by the loss of GSH binding ca-

pacity. To determine whether this change shows in the

overall shape of the fibrils, untreated and heat-treated

Ure2p fibrils were observed in the electron microscope

following negative staining. The two kinds of fibrils were

found indistinguishable (Figs. 1B and C). Both kinds of

fibrils are 20 nm wide and over 1 lm long. We con-clude from this observation that the conformational

rearrangement that leads to the loss of ligand binding

capacity of Ure2p does not perturb the overall mor-

phology of the fibrils. We further conclude that heat

treatment of the native-like assembled form of Ure2p

in solution is not sufficient to disrupt their fibrillar

structure.

3.2. The proteolytic patterns of untreated and heat-treated

Ure2p fibrils differ significantly

Using proteinase K treatment, we previously showed

that the time courses of degradation of the soluble and

assembled forms of Ure2p differ significantly. Interest-

ingly however the patterns show no significant differ-

ences in the polypeptide species generated which suggestthat the overall conformation of Ure2p is not altered

upon assembly into fibrils (Thual et al., 1999). The

degradation pattern of soluble and assembled Ure2p

was further shown to be indistinguishable from that of

Ure2p 95–354 indicating that the polypeptide species

generated upon proteinase K treatment are derived from

the C-terminal domain of the protein (Thual et al.,

2001). Proteinase K treatment is thus an excellent toolfor probing conformational changes that occurs in the

compactly folded, mainly a-helical C-terminal domain

of Ure2p. Fig. 2 shows the degradation products of

untreated and heat-treated Ure2p fibrils. In native-like

fibrils, full-length Ure2p is degraded within 2min into a

polypeptide with a molecular mass of 30 kDa that cor-

responds to Ure2p 95–354. Six other polypeptides with

apparent molecular masses 29, 24, 22, 20, 19, and14 kDa are generated. Three polypeptides resist the

treatment for over 30min (29, 20, and 14 kDa). They are

degraded 60min after the onset of the cleavage reaction.

Full-length Ure2p resists proteinase K treatment for

over 5min in heat-treated fibrils. Its degradation at early

times following the onset of proteinase K treatment

generate a cluster of polypeptides that have apparent

molecular masses in the range 34–27 kDa. These trun-

cated Ure2p polypeptides represent over 90% of the

degradation products. Minor polypeptides with appar-

ent molecular masses between 26 and 20 kDa are also

generated. At later stages of the degradation reaction

three polypeptides with apparent molecular masses 30,29, and 23 kDa that differ from those generated from

untreated fibrils are observed. We conclude from these

observations that a major change in the conformation of

Ure2p in the fibrils occurs during heating with the loss

or protection of several proteinase K cleavage sites

probably due to increased packing of Ure2p molecules

within the fibrils.

3.3. XRD reveals different molecular architectures for

native-like and heat-treated Ure2p fibrils

Examination of the X-ray scattering patterns from

heat-treated, Fig. 3A, and native-like fibrils Fig. 3B,

reveals changes in the molecular organization of Ure2p

within the fibrils.

The pattern in Fig. 3A resembles the classical patternof others amyloid fibrils, with the characteristic cross-bsheet reflection at 4:71 0:01�AA spacing, the rather

broad ring around 10�AA, and another reflection at

3:83 0:02�AA. The pattern is anisotropic, the 4.71 and

3.83�AA rings are slightly reinforced along a direction

which can be assumed to be the fibrils direction (me-

ridian). The intensity profile of the 4.71�AA reflection is

regular and unlikely to be due to the overlap of the re-flections of several structural features. More interesting,

the 10�AA ring is also anisotropic, its maximum along the

meridian and the equator are respectively located at

10:05 0:05 and 10:45 0:05�AA. This clearly indicates

that the ‘‘10 �AA’’ ring originates from two different

structural features, the cross b intersheet distance gives

rise to the equatorial reflection, whilst the origin of the

meridional reflection is unclear. It could come from an

Fig. 2. The degradation patterns of native-like and heat-treated Ure2p

fibrils are unrelated. Native-like (A) and heat-treated (B) Ure2p fibrils

were subjected to proteinase K treatment, and the time courses of the

digestions monitored by SDS–PAGE (15%) followed by immunode-

tection using an anti-Ure2p polyclonal antibody. Time points (in

minutes) are shown at the top of each gel. Molecular mass markers (in

kDa) and the migration front (MF) of the gel are shown on the left.


average distance between the chains issued from the

neighboring strands within a given cross-b sheet. A

similar anisotropic b-sheet ring has already been ob-

served for keratin fibers in stratum corneum corneocytes

(Garson et al., 1991). These features are compatible

with orthorhombic cross-b sheet crystallites having

cell dimensions a ¼ 9:42 0:2�AA, b ¼ 6:6 0:5�AA and

c � 10:05�AA, where a, refers to the fibril axis (hydrogen-bonding direction), b, to the polypeptide chain direc-

tion, and c, to inter-sheet direction. The value of b was

deduced from the 3.83 and 4.71�AA reflections. These re-

flections were attributed Miller�s indexes (2 1 0) and

(2 0 0), respectively. As for the previous works on amy-

loid fibrils, which all lead essentially to the same unit cell

parameters, no information concerning the parallel

versus anti-parallel character of the b-sheet foldingcould be determined. We estimated the size (coherent

length) L of the crystallites in the three directions a, b,

and c, from the angular widths of the reflections using

the formula:

L ¼ 1=ðD2exp � D2

instÞ1=2, where Dexp and Dinst are the

experimental and instrumental full width at half maxi-

mum. The 4.71�AA reflection gave a size of 80�AA along a

direction, and the equatorial 10.05�AA reflection about30�AA along c direction. The size of the crystallite along

the b direction (about 25�AA) was derived from the an-

gular width of the (2 1 0) reflection (3.83�AA). It was as-

sumed that its ellipsoidal shape in the reciprocal space

can be modeled by the product of the (2 0 0) and (0 1 0)

radial profiles, taking into account the disorientation

effect. Our crystallite sizes along a and c are similar to

those observed for other amyloid fibrils (Kirschner et

al., 1986). So far the crystallite size along direction b has

never been calculated along the b-strand. The structure

of dehydrated microcrystals of a synthetic peptide re-

producing an N-terminal fragment of the yeast prion

Sup35p has been interpreted as a compact packing of

cross-b sheets with a b parameter equal to 39.4�AA (Bal-birnie et al., 2001). This is compatible with the coherent

length we measure along direction b.

The X-ray pattern from native-like fibrils presented in

Fig. 3B differs significantly from that of heat-treated

fibrils (Fig. 3A). The characteristic 4.7 and 3.83�AA re-

flections are missing and the wide-angle scattering re-

gion only contains a very broad ring centered at 4.3�AA.

We did not find in the literature any reference to X-raypattern of prion or amyloid fibrils devoid of the char-

acteristic cross-b sheet signal. The 10�AA ring is present, it

is isotropic, centered at 10.5 �AA and its width corresponds

to a coherent length of about 30�AA. On the contrary, the

small-angle region (insert in Fig. 3B) displays several

scattering features that are not observed for the heat-

treated fibrils (insert in Fig. 3A). The most prominent

one is an arc at 47�AA, which is sharp enough to corre-spond to a coherent length larger than 500�AA. This re-

flection indicates that native-like fibrils are well oriented

in the capillary. Two broad rings are observed: a rather

strong one at 52�AA and a very weak one at 25�AA. A broad

streak in the inner part of the pattern is typical of the

equatorial scattering by cylinder-shaped objects, the

Fig. 3. X-ray diffraction patterns of native-like and heat-treated Ure2p fibrils obtained on station ID14-EH1 (ESRF-Grenoble, France) on con-

centrated samples (see text) using a 50lm diameter X-ray beam. (A) heat-treated fibrils. The pattern shows the characteristic 4.7�AA thin arc along the

meridian (axis) that is characteristic of amyloid fibrils. No additional scattering features in the central part of the pattern (i.e., in the range 30–80�AA

periodicity) are visible (see insert). (B) native-like fibrils. The 4.7�AA arc is absent, scattering features are visible in the central part of the pattern (see

insert), in particular a strong arc that corresponds to a 47�AA axial periodicity.


fibrils in this case. Therefore, the 47�AA sharp arc is me-

ridional and reveals the existence of a long-range peri-

odicity along the fibrils. The broadness of the 52 and25�AA rings indicate that these features probably arise

from lateral intra-fibril interferences between protofibril

subunits. We conclude from these observations that

native-like and heat-treated fibrils possess each a char-

acteristic architecture. The two architectures are dis-

tinct. This reflects two different spatial organizations ofUre2p molecules within the two kinds of fibrils. We

further conclude that the characteristics of native-like

fibrils differ significantly from that of authentic amy-

loids.

3.4. Native-like and heat-treated Ure2p fibrils differ by

their secondary structural content

The peaks observed on the second derivative of the

FTIR spectra of native-like, heat-treated Ure2p fibrils

and crystals of Ure2p 95-354 were used for fitting the

respective FTIR spectra. The data are presented in Fig. 4

and Table 1. The a-helical (33%) and b sheet (21%)

contents of Ure2p 95–354 crystals are consistent with

those obtained using Raman spectroscopy for the full-

length protein (Taylor et al., 1999). The measured sec-ondary structure content differs significantly from that

calculated from the crystal structure of Ure2p 95–354

(58% a-helices and 7% b-sheets) (Bousset et al., 2001a).

The difference might come from a change in the con-

formation of Ure2p 95–354 upon washing the pre-

cipitant away by addition of water droplets to the

crystals, mopping up the droplets using filter papers and

drying the crystals prior to FTIR measurements.Comparison of the data obtained for native-like and

heat-treated Ure2p fibrils to that of the crystal form of

Ure2p 95–354 revealed an increase in both the a helical

and b sheet contents. The a helical content increased

from 33% in the crystal form of Ure2p 95–354 to 36 and

40% in heat-treated and native-like Ure2p fibrils, re-

spectively, while that of b sheets increased from 21% to

46 and 28%, respectively. These observations are con-sistent with a decrease in flexible regions and an increase

in structured regions upon assembly of Ure2p into fibrils

due in part to the stabilization of the poorly structured

N-terminal region of the molecule in a defined confor-

mation. Were the N-terminal 94 residues out of 354

amino acids of Ure2p to convert to a b-sheet during

assembly under physiologically relevant conditions, a

26% increase in b-sheet content would be observed. Themeasured increase in b-sheet content of native-like fibrils

is of 7%, incompatible with the conversion of the N-

terminal 94 residues into a b-sheet. The extent of sec-

ondary structure rearrangement that accompanies

Ure2p assembly into fibrils under physiologically rele-

vant conditions as measured by FTIR spectroscopy

Fig. 4. Native-like and heat-treated fibrils differ by their secondary

structural contents. FTIR spectra (bold line) of fibrils assembled under

physiologically relevant conditions from full-length Ure2p (A), Heat-

treated Ure2p fibrils (B) and crystals of Ure2p 95–354 (C). Curve fit

spectra (thin lines) are presented in each case.

Table 1

Ure2p secondary structure contents determined by deconvolution of the FTIR spectra for native-like fibrils, heat-treated fibrils, and Ure2p 95–354

crystals presented in Fig. 4

a-Helix content (%) b-Sheet content (%) Turn/bend content (%) Coil content (%)

Native-like fibrils 40 28 11 20

Heat-treated fibrils 36 46 10 7

Ure2p 95–354 crystals 33 21 24 22


further confirm the lack of cross b-core in native-likefibrils as observed in X-ray fiber diffraction images. We

conclude, therefore that the fibrils do not comprise a

cross b-core that is typical of amyloids. In contrast, heat

treatment of Ure2p fibrils increases significantly (65%)

their b sheet content with the concomitant appearance

of the 4.7�AA band in X-ray fiber diffraction images typ-

ical of amyloids.

4. Discussion

The data presented above further support our recent

findings that the fibrils assembled from full-length

Ure2p under physiologically relevant conditions despite

having many properties akin to amyloid such as an in-

creased resistance to proteolysis and yellow-green bire-fringence upon Congo Red binding are devoid of the

amyloid characteristic cross b-sheet core (Bousset et al.,

2002). When native-like fibrils are heated, a major

conformational change occurs with the constitution of a

cross b-sheet core typical of amyloid fibrils. This rear-

rangement is accompanied by a major conformational

change in Ure2p with the loss of the ability to bind

GSH, a change in the proteolytic pattern and an in-crease in b-sheet content in the fibrils. The transition

from a helical polymer made of native-like polypeptide

chains to a cross-b structure made of non-native mole-

cules does not reveal in a change in fibril morphology.

Indeed, electron micrographs of untreated and heat

treated fibrils are indistinguishable.1 This transition

shows however in X-ray diffraction patterns and FTIR

measurements.A plausible low-resolution structural model for na-

tive-like and heat-treated Ure2p fibrils needs to account

for EM, FT-IR, and XRD observations. Native-like fi-

brils contain no cross-b core. Their longitudinal orga-

nization is dominated by a 47�AA long-range axial repeat

that does not arise from a helical coil but probably from

the constituting Ure2p molecules since it corresponds

with one of the dimensions of Ure2p. Indeed, the vol-ume of hydrated Ure2p monomer derived from the

crystal structure of Ure2p 95–354 (Bousset et al., 2001a)

is 69:2� 27:8� 52:1�AA3

while that of the dimer is

83:1� 50:4� 53:6�AA3. Native-like fibrils are built either

of monomeric or dimeric molecules associated in a non-

native manner (Bousset et al., 2002). The 47�AA repeat is

compatible with either one of the dimensions of the

monomer or with that of the dimer. Furthermore, thepresence of equatorial broad reflections at 52 and 25 �AAsuggest that Ure2p fibrils could be constituted by the

lateral association of protofibrils that are about 50 �AA in

diameter. The scattered intensity IðSÞ by one fibril could

be modeled as: IðSÞ ¼ ZðSÞ:jF ðSÞj2, where ZðSÞ is the

interference function, i.e. the Fourier transform of the

distribution function of the protofibrils within the fibril,

and F ðSÞ is the Fourier transform of the electron densityof the protofibril section. If we assume that the pro-

tofibrils are cylindrical in shape (with a radius r),

F ðSÞ / r2J1ðuÞ=u, with u ¼ 2prS. The scattering inten-

sity assuming a square lattice with unit-cell length equal

to 50�AA leads to scattering peaks close to 50 and 25 �AA.

Heat-treated Ure2p fibrils although indistinguishable

from native-like fibrils in the electron microscope have

lost the axial 47�AA repeat that characterizes native-likefibrils. Thus heat treatment yields fibrils devoid of the

native-like fibril characteristic axial repeat. These fibrils

show instead the typical cross b-sheet core of amyloids.

The radial size of the b-sheet core is 30�AA. Strikingly

however and in contrast to models of amyloid fibrils

made of transthyretin (Blake and Serpell, 1996) and the

SH3 domain of bovine phosphatidyl-inositol kinase

p85a subunit (Jimenez et al., 1999), Ure2p fibrils exhibita rather small coherence length (80�AA) which indicates

that their b-core is short ranged.

Although the coherence length is small, it is reason-

able to consider that the fibrils are made of a continuous

cross-b core running along their axis. The limited

number of scattering features in the XRD pattern indi-

cate that heat-treated Ure2p fibrils differ from other

amyloid fibrils (Blake and Serpell, 1996; Jimenez et al.,1999) in that they show no helical axial repeat.

The 30�AA b-sheet core is due to a conformational

change either within a protofibril or at the interface be-

tween two adjacent protofibrils. Given that the overall b-sheet content of the fibrils is 50%, the surface area that is

not involved in the b core must represent the remaining

50% of the overall surface in a fibril cross-section. A

scheme summarizing these parameters is shown in Fig. 5.Interestingly, the diameter of these protofibrils remains

similar to that made of native-like polypeptide chains,

thus accounting for the absence of measurable change in

fibril width as determined by electron microscopy.

4.1. Biological significance

Yeast prions assemble in vitro in a catalytic mannerinto fibrils. The limiting step in the assembly reaction is

the nucleation step (Glover et al., 1997; Thual et al.,

1999). This is strongly in favor of a polymerization

model in which prion molecules assemble in a manner

similar to the crystallization of a solute molecule in so-

lution. In such a process the energetically unfavorable

nucleation step is followed by a more favorable elon-

1 The coherence length calculated from the X-ray diffraction

patterns is not readily comparable to the persistence length of the

fibrils that can be derived from electron micrographs. Indeed, the loss

of regular periodicity does not necessarily show in a change in the

rigidity of a fibril. It is therefore not unusual to observe similar

morphologies for native-like and heat-treated fibrils at the electron

microscope resolution used in this study.


gation reaction that proceeds in a closed system until

equilibrium is reached between unassembled and poly-

meric species. It is reasonable to envisage that Ure2p has

the ability to assemble into different polymers in a

manner similar to the crystallization of a large number

of proteins in different crystal lattices. Although thepresent work does not bring evidence for such a process,

we clearly show that a major conformational change

occurs upon heating the fibrils. Such a change is similar

to that observed upon heating protein polymers (Ast-

bury and Woods, 1933).

The finding that native-like and heat-treated Ure2p

fibrils are indistinguishable in the electron microscope

although they differ both in their overall secondarystructure content and their intrinsic supramolecular or-

ganization suggests that the fibrils could be made of the

arrangement of Ure2p polypeptide chains in at least two

manners. Such different arrangements should reflect in

the elongation properties of the fibrils since the geome-

try of polymers ends regulates their growth rate by

favoring or unfavoring intermolecular interactions be-

tween soluble Ure2p polypeptide chains and fibrils ends.Alternatively, the growth rate of the two kinds of fibrils

may be regulated by the higher or lower propensity of

defined Ure2p unfolding intermediates to interact with

fibril ends. Finally, such different arrangements are

expected to imprint a specific orientation to Ure2pmolecules upon incorporation at fibrils ends leading to

the propagation of a defined structural motif that could

account for the experimental differences in conver-

sion efficiencies at the origin of the strain concept in

prion propagation (Schlumpberger et al., 2001; Uptain

et al., 2001).

To determine whether the two types of Ure2p fibrils

can propagate their respective structural characteristicsduring the construction of the fibrils by either selecting

Ure2p molecules in different conformations or imprint-

ing a specific orientation or conformation to newly in-

corporated Ure2p molecules, native-like seeds and heat

treated seeds were diluted in the presence of soluble

Ure2p and the structure of newly assembled fibrils as-

sessed by proteinase K treatment. Ure2p was found to

assemble into the native-like type of fibrils when seededeither by native-like or heat-treated fibrils (not shown).

This result either indicates that heat-treated fibrils ends

are indistinguishable structurally from that of native-

like fibrils or that they are unable to elongate. To dis-

tinguish between the two possibilities, Ure2p assembly

kinetics were measured following seeding by the two

kinds of fibrils. The data are shown in Fig. 6. The as-

sembly of Ure2p in the presence of native-like fibrils wasfound to proceed without a lag phase in accord with the

limiting nucleation phase being bypassed. In contrast, a

lag phase precedes the elongation phase in Ure2p as-

sembly reactions in the presence of heat-treated fibrils

indicating de novo nucleation of native-like Ure2p fibrils.

Thus, Ure2p does neither interact with heat-treated fibril

ends nor incorporate into such preformed fibrils.

We recently proposed by analogy to the mechanismof assembly of serpins into protein fibrils (Carrell and

Gooptu, 1998) two scenarios that account for the lim-

ited structural rearrangement at the origin of Ure2p

assembly under physiologically relevant conditions

Fig. 6. Native like fibrils seed Ure2p assembly into protein fibrils, while

heat-treated fibrils do not. Time courses of Ure2p (170lM) assembly

into protein fibrils in 50mM Tris, pH 7.5, 100mM KCl, in the absence

of added nuclei (d, dotted line), upon addition of 5.5lM pre-assem-

bled native-like (j) or 5.5 lM heat-treated (m, plain line) Ure2p fibrils.

The assembly of Ure2p into protein fibrils was monitored by Thio-

flavin-T binding.

Fig. 5. Schematic representation of a cross b-sheet core indicating the

dimensions of the unit cell and the domain size of heat-treated Ure2p

fibres. The folding elements that spread from the core are purely hy-

pothetical. They are represented to underline the existence of non b-sheet parts representing about 50% of the molecules that are located

outside the core.


(Bousset et al., 2002). Other scenarios where b-sheetsorganized in helices running along the core of the fibrils

have been recently proposed for the assembly of syn-

thetic polypeptides into amyloid fibrils (Perutz et al.,

2002; Wille et al., 2002). In these models, the b-sheetcore of the fibrils is therefore expected to resist prote-

olysis. These scenarios do not apply for the assembly of

full-length Ure2p into fibrils under physiologically rel-

evant conditions as the degradation patterns of solubleand assembled Ure2p generate identical polypeptides

(Thual et al., 1999) and the fibrils lack the 4.7�AA reflec-

tion that characterizes amyloids. The finding that heat-

treatment of native like Ure2p fibrils generates b-sheetrich fibrils indicates that parts of Ure2p molecules in the

fibrils are flexible and that the heat-induced structural

rearrangement leads to the reorganization within the

fibrils of Ure2p flexible parts into anti-parallel b-sheetthat may be organized in helices. Ure2p N-terminal

domain and Ure2p 94–200 fragment that contains a b-sheet (Bousset et al., 2001a) have the highest probability

to be involved in such a transition. Thus, future efforts

to identify Ure2p regions that are at the origin of the a-helical to b-sheet transition would allow a better com-

prehension of the flexibility of Ure2p within the fibrils.

Amyloid formation is believed to be a generic prop-erty of polypeptide chains. It is also believed that the

form of a given protein that assembles into amyloid fi-

bers is a partially unfolded polypeptide chain. The fact

that only a subset of proteins possess the capacity to

form such polymers under non-denaturing conditions

has been attributed to an insufficient amount of folding

intermediate that plays the role of amyloid precursor

(Dobson, 1999). To populate the folding intermediatesthat are precursors of amyloid fibrils it is common to

incubate various polypeptide chains at high tempera-

tures and/or extreme pHs (Jimenez et al., 2002). The

transition we report here differs significantly from the

partial unfolding of a polypeptide chain in solution and

its consequent assembly into amyloid fibrils as the a-helical to b-sheet transition occurs within the fibrils.

Finally, the ability of Ure2p fibrils obtained underphysiological conditions to evolve in vivo into the b-sheet rich fibrils remains to be determined and the

function of such an evolution elucidated. It is important

to bear in mind though that while native-like fibrils as-

semble in a nucleation-dependent manner, the nucle-

ation phase being abolished upon addition of minute

amounts of preformed fibrils to soluble Ure2p (Thual et

al., 1999), a property central to prion propagation(Bousset and Melki, 2002), heat-treated fibrils do not

meet with this property. This strongly suggests that b-sheet rich fibrils if formed in vivo are inert end products.

In the yeast cytosol, Ure2p fibrils appear to be packed

into very dense, high molecular weight particles (Fer-

nandez-Bellot et al., 2000) that may correspond to su-

pramolecular assemblies. This packing is either the

consequence of fibrils ageing in a particular physico-chemical microenvironment or due to the effect of pro-

teins associated to Ure2p fibrils. It is reasonable to

envisage that such a packing could be the consequence

of a conformational change within the fibrils. In prin-

ciple, Ure2p fibrils in such bundles must be well oriented

and therefore suitable for X-ray fiber diffraction mea-

surements. Partner proteins associated to the fibrils may

affect the intrinsic structure of the fibrils. Alternatively,they may produce an additional signal that superim-

poses to that of the fibrils X-ray diffraction patterns.

Thus, future efforts to isolate fibril bundles free of as-

sociated proteins for X-ray diffraction studies must be

attempted. It would also be very interesting to carry out

such studies on fibrils with associated proteins.

Acknowledgments

We gratefully acknowledge the expert help of Hassan

Belrhali on beamline ID14-EH1 in data collection and

Paul Dumas for his help in FTIR measurements. Wethank Dr. Solange Morera for allowing us to meet and

initiate our collaboration and Dr. Virginie Redeker and

Sheena Radford for helpful discussions. This work was

supported by the French Ministry of Research and

Technology, the Centre National de la Recherche Sci-

entifique and the Association pour la Recherche sur le

Cancer.

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