Assembly of the Asparagine and Glutamine Rich Yeast Prions into Protein Fibrils

Current Alzheimer Research, 2008, 5, 251-259
251
Assembly of the Asparagine- and Glutamine-Rich Yeast Prions into Pro-
tein Fibrils
Luc Bousset, Jimmy Savistchenko and Ronald Melki*
Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
Abstract: The proteins Ure2, Sup35 and Rnq1 from the baker’s yeast have infectious properties, termed prions, at the ori-
gin of heritable and transmissible phenotypic changes. It is widely believed that prion properties arise from the assembly
of Ure2p, Sup35p and Rnq1p into insoluble fibrils.
Yeast prions possess regions crucial for their propagation that can be either N- or C-terminal. These regions have unusual
amino acid composition. They are very rich in glutamine and asparagine residues and resemble in that to huntingtin, a pro-
tein involved in the neurodegenerative Huntington’s disease.
Yeast prions assembly process has been hypothesized to be the consequence of the properties of glutamines and asparagi-
nes to engage in polar protein-protein interactions, termed polar-zippers. While this can certainly occur under certain con-
ditions, glutamine and asparagine residues can establish other kinds of interactions with a variety of amino acid residues
thus mediating protein-protein interactions involved in the assembly of polypeptide chains into high molecular weight oli-
gomers.
This review details the interactions that can be established by glutamine and asparagine residues that may allow a better
understanding of their role in mediating protein-protein interactions and prion propagation.
Keywords: Prion, [PSI+], [URE3], Sup35p, Ure2p, amyloid fibrils, native-like assemblies,
INTRODUCTION
appear as a consequence red, while [PSI+] cells are either
white or of different pink shades. The overexpression of
In the yeast Saccharomyces cerevisiae, three traits,
Sup35p increases the frequency of [PSI+] appearance. This is
[PSI+], [URE3] and [PIN+] are inherited in a non-Mendelian
also what Rnq1p does. Rnq1p has therefore a “[PSI+] induc-
manner [1-3]. These three epigenetic factors are due to the
ing” activity heritable in a non-mendelian manner termed
prion properties of the proteins Sup35, Ure2 and Rnq1, re-
[PIN+].
spectively. The eukaryotic release factor eRF3 or Sup35p
mediates together with eRF1 or Sup45p the termination of
THE [URE3] TRAIT
protein translation in S. cerevisiae [4-6]. The exact functions
of Ure2p and Rnq1p are unknown. Ure2p acts as a negative

Yeast cells grown in the presence of simple nitrogen
regulator of nitrogen metabolism [7-9] while Rnq1p modu-
sources such as ammonia turn off the synthesis of a number
lates [PSI+] appearance [3, 10]. In the prion states [PSI+],
of enzymes and transporters needed for the assimilation of
[URE3] and [PIN+], insoluble aggregates of Sup35p, Ure2p
complex nitrogen sources. This is believed to be the conse-
and Rnq1p, respectively, are observed [11-16].
quence of the cytoplasmic interaction of Ure2p with a tran-
scription factor named Gln3p. The interaction of soluble
THE [PSI+] AND [PIN+] TRAITS
Ure2p with Gln3p prevents the entry of the latter protein into
the nucleus and the transcription of unnecessary genes [17-

As translation termination is altered in [PSI+] cells fol-
18]. In [URE3] cells Ure2p aggregates in the cytoplasm.
lowing Sup35p aggregation, the ribosomes have an increased
Gln3p is therefore no more sequestered in the cytoplasm and
tendency to read through nonsense stop codons. Thus, while
the translation of genes involved in complex nitrogen
wild type cells ([psi-]) carrying an ochre (UAA) stop codon
sources catabolism becomes constitutive even when the cells
in the ADE2 gene involved in adenine synthesis, a truncated,
are grown in media containing simple nitrogen sources lead-
non functional form of the enzyme (phosphoribosyl amino-
ing to an altered nitrogen metabolism.
imidazole carboxylase) is synthesized, in [PSI+] cells, a frac-
tion of ADE2 gene products are of the correct length and
THE PROTEINS Sup35, Rnq1 AND Ure2 FROM S. cer-
functional. The interruption of adenine synthesis cycle leads
evisiae
to the accumulation of a colored precursor of adenine (5’-

Sup35p (Swiss-Prot P05453) is a large 685 amino acid
Phosphoribosyl-5-aminoimidazole) in wild type cells which
residues polypeptide with a calculated molecular mass of

76551Da [19]. One can distinguish three regions in Sup35p
*Address correspondence to this author at the Laboratoire d’Enzymologie et
(Fig. 1). The N-terminal region (N) extends from amino acid
Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-
residues 1 to 123 is rich in Q, N and G residues (47%). This
Yvette, France; Tel: 33 169823503; Fax: 33169823129;
region is not essential for the role of the protein in translation
E-mail: melki@lebs.cnrs-gif.fr
termination [20-22]. It is called prion domain as it plays a

1567-2050/08 $55.00+.00
©2008 Bentham Science Publishers Ltd.

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252 Current Alzheimer Research, 2008, Vol. 5, No. 3
Bousset et al.
critical role in prion propagation. The middle region (M) has

Ure2p (Swiss-Prot P23202) has a calculated molecular
probably a structural function. It extends from amino acid
mass of 40271 Da and is composed of 354 amino acid resi-
residues 124 to 253. The C-terminal region is the functional
dues (Fig. 1). Ure2p is a two-domain protein. The N-terminal
domain of the protein providing translation-termination ac-
part, required for the propagation of the [URE3] phenotype
tivity [5, 20]. It is compactly folded based on the crystal
[14], extends from amino acid residue 1 to 93 [24]. This do-
structure of eRF3 from S. pombe [23] and contains the GTP
main is rich in Q, N, S and T residues (62%) and flexible
and eRF1-binding sites (Fig. 1; PDB accession code 1R5N).
[25]. The C-terminal domain that complements URE2 gene
deletion [14] extends from amino acid residues 94 to 354

Rnq1p (Swiss-Prot P25367) a 42.5kDa polypeptide is
[24] is compactly folded [26, 27] (Fig. 1; PDB accession
unusually rich in Q and N residues in particular in its part
code 1G6Y), binds glutathione [28] (PDB accession code
extending from amino-acid residue 153 to 405 (Fig. 1) [16].
1JZR) and exhibits glutathione perroxidase activity [29].
This Q- and N-rich C-terminal domain is crucial for [PIN+]
propagation.















Fig. (1). Primary structures of Sup35pNM, Ure2p1-93 and Rnq1p153-405. Q residues are colored red, N are colored blue, G, S and T are
colored green. The M domain of Sup35pNM is underlined. The structure of the compactly folded N-terminal domains of Sup35p from S.
pombe and that of Ure2p from S. cerevisiae are shown. The three subdomains of Sup35p C-terminal domains are colored blue, red and green
and the peptide that links this domain to the M domain of the protein is colored purple. The two subdomains of Ure2p C-terminal domain are
colored blue and green. The letters N and C in the structure panels refer to the N and C-terminal residues of the polypeptide represented. The
GTP molecule bound to Sup35p is colored in red. The structures were generated with PyMol [http://pymol.sourceforge.net/]. The PDB coor-
dinates used for the panels A and B are1R5N and 1G6Y, respectively.

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Assembly of the Asparagine- and Glutamine-Rich Yeast Prions
Current Alzheimer Research, 2008, Vol. 5, No. 3 253
ASSEMBLY OF Sup35p, Rnq1p AND Ure2P INTO
red and Thioflavin T and exhibit yellow-green birefringence
PROTEIN FIBRILS
in polarized light upon Congo red binding [25, 30]. These
characteristics together with the findings that the N-terminal

Purified full-length Sup35p, Rnq1p and Ure2p assemble
domains of Sup35p and Ure2p that are critical for prion
at neutral pH and under physiological salt concentrations
propagation, assemble in vitro into fibrils that exhibit a 4.7 Å
into high molecular weight oligomers. These oligomers are
reflection in X-fiber diffraction images [33-34] led to the
either soluble and spherical or insoluble and fibrillar (Fig. 2)
view that full-length Sup35p and Ure2p fibrils are conven-
[24, 30-32]. The formation of the unbranched fibrils is
tional amyloids.
greatly accelerated by seeding with preformed fibrils sug-
gesting that the limiting step in the assembly process is nu-
DEFINITION OF AMYLOIDS
cleation.
The term amyloid signifies starchlike. This term was

used for the first time in the 19th century to refer to deposits

within the brain that stain pale blue with iodine and violet

upon subsequent addition of sulfuric acid as does starch de-
posits in plants. The term amyloid was maintained later on

when it was established that these deposits lack carbohy-

drates and are made of proteins [35].


The Nomenclature Committee of the International Soci-
ety of Amyloidosis has defined the term amyloid as “ex-

tracellular depositions of protein fibrils with characteristic

appearance in electron microscope, typical X-ray diffraction
pattern and affinity for Congo red with concomitant green

birefringence” [36-37]. The committee also stated that “in-

tracytoplasmic and intranuclear protein aggregates with
some similarity with amyloid, should be separately compiled

as a list of close relatives of amyloid proteins” [37]. The sci-

entists involved in the characterization at the molecular level
of amyloid deposits have defined structurally amyloid fibrils.

The structure-based definition does not require an extracellu-

lar localization and includes intracellular protein fibrils asso-

ciated with disease. Amyloid fibrils bind the dyes Congo red
and thioflavin T. However, the binding mechanism of these

dyes, in particular Congo red that has been used as early as

in 1927 [38], to the fibrils is i- not well understood ii- highly
dependent on the staining conditions [39-40] and iii- not

specific [41], this reaction is therefore not suitable to define

amyloid fibrils at the structural level. Amyloid fibrils are
therefore defined as fibrillar polypeptide aggregates with

cross- conformation i.e. a structure where the hydrogen

bonds between two consecutive sheets are oriented parallel
to the main fibril axis while the constituting -strands are

oriented transversely to the main fibril axis [42-43]. This

type of structure gives rise to a characteristic pattern of re-
flexions in X-ray diffraction experiments. This pattern con-

sists of a conserved 4.6-4.8 Å meridional spacing and an

equatorial spacing of about 10 Å (Fig. 3). The 4.6-4.8 Å re-

flexion comes from the distance between two hydrogen
bonded strands and is invariant as it depends on the geome-

try of the polypeptide backbone. It is referred to as the “main

chain spacing”. The equatorial reflection at about 10 Å
comes from the packing distance between two juxtaposed -
Fig. (2). Electron micrographs of negatively stained samples of A,
sheets [44-45]. This distance can vary with the amino acid
Ure2p soluble oligomers, B, Ure2p fibrils, C, Sup35p soluble oli-
composition of the polypeptide as it depends on the orthogo-
gomers, D, Sup35p fibrils. The bars represent 0.1 m in A and B,
nal protrusion of the amino acid side chains from the plane
0.2 m in C and D.

of the sheet. It is worth noting that this reflection is not ob-

Aside being unbranched, fibrils formed from full-length
served when the inter-sheet spacing is not regular. Finally, it
Sup35p and Ure2p exhibit a number of characteristics of
is worth mentioning that it is not yet clear whether the sheets
amyloids. They are about 20nm wide and over 1 m long [24,
that constitute amyloid fibrils are parallel, antiparallel or
30-31], have increased resistance to proteolysis as compared
mixed.
to the soluble proteins [24, 30]. They bind the dyes Congo

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254 Current Alzheimer Research, 2008, Vol. 5, No. 3
Bousset et al.







Fig. (3). Schematic representation of the packing of the -strands within an amyloid fibril and of the X-ray diffraction pattern such packing
generates. The inter-strand (4.7Å) and inter-sheet (10Å) distances are indicated. The orientation of the strands and sheets relative to the fibril
main axis is also shown.
Cross- structures have also a characteristic signature in
in flexible regions of the protein as in the case of actin fila-
Fourier-transform infrared spectroscopy. Their -sheet con-
ments [51], microtubules [52], intermediate filaments [53],
tent gives rise to an amide I maximum at 1610-1630 cm-1
flagellins [54] and sickle-cell hemoglobin [55] assemblies or
which allows distinguishing them from sheets present in
domain swapping as in the case of 1-antitrypsin [56] PrP
soluble proteins and in assemblies made of native globular
[57] and ribonuclease [58] assemblies. The structural charac-
polypeptides [46-47].
terization of the fibrillar form of full-length Ure2p assembled
under physiological conditions indicates that the fibrils are

It is worth noting that the vast majority of polypeptide,
made of native-like functional units in a manner reminiscent
including -helical proteins, can form amyloid fibrils in vitro
of the assembly of the abovementioned polypeptides.
depending on the experimental conditions used. Amyloid
formation is therefore a generic property of polypeptides
COMPARISON OF THE ASSEMBLY PROPERTIES
[48].
OF FULL-LENGTH Sup35p AND Ure2p AND THEIR
N-TERMINAL DOMAINS
STRUCTURAL CHARACTERIZATION OF THE
FIBRILLAR STATES OF FULL-LENGTH Sup35p
The critical concentrations of full-length Sup35p and
AND Ure2p UNDER PHYSIOLOGICAL CONDITIONS
Sup35pNM are of the same order of magnitude and the two
polypeptides co-assemble indicating that they share struc-
While full-length Sup35p fibrils have not been fully
tural similarities [31]. However, the number of polypeptide
characterized structurally, full-length Ure2p fibrils appear to
chains in Sup35p and Sup35pNM nuclei, the assembly and
have several characteristics that set them apart from classical
elongation rates of the two polypeptides and the efficiency of
amyloids. Limited proteolysis patterns of the soluble and
de novo induction of [PSI+] appearance upon reintroduction
fibrillar forms of the protein are very similar [24, 41]. Fou-
within the yeast cell of the two kinds of fibrils made in vitro
rier-transform infrared spectroscopy (FTIR) showed a very
differ significantly, suggesting structural diversity [31].
slight structural change between soluble and fibrillar Ure2p
and revealed that both contained a large proportion of -
The assembly of Ure2p1-93 and full-length Ure2p at
helical structure [49]. Furthermore, partially aligned Ure2p
identical concentration differ significantly as observed upon
fibrils give X-ray fiber diffraction patterns that are inconsis-
comparison of the assembly kinetics of a Ure2p variant con-
tent with cross- structure. Such patterns consist of low-
taining a Factor Xa specific cleavage site located between
angle diffraction signals at 47 Å on the meridian and 25 Å
the N- and C-terminal domains of the protein in the presence
and 52 Å on the equator [50], consistent with the regular
and the absence of the protease. The lag phases preceding
packing of globular Ure2p molecules which dimension, de-
assembly and the extent of thioflavin T binding differ sug-
termined from the three-dimensional structure of the protein,
gesting that the fibrils are different (Fig. 4a). This is indeed
is 70 Å x 28 Å x 52 Å. In addition, Ure2p within the fibrils
the case. Full-length Ure2p fibrils are 20nm wide while
binds glutathione [28] and exhibits glutathione perroxidase
Ure2p1-93 fibrils are about 4 nm wide (Fig. 4c and d). Inter-
activity [29] as does the native, soluble form of the protein.
estingly however, when the generation and assembly reac-
tions of Ure2p1-93 are compared, it appears that although

A number of polypeptide assemble into fibrils without
cleavage by the protease Factor Xa has gone to completion
any loss of native structural elements. The assembly of such
within 5 hours as shown by SDS-PAGE analysis (Fig. 4b),
polypeptides does not exclude limited structural adjustment

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Assembly of the Asparagine- and Glutamine-Rich Yeast Prions
Current Alzheimer Research, 2008, Vol. 5, No. 3 255

























Fig. (4). Assembly of full-length Ure2p and Ure2p1-93. A, time courses of full-length Ure2p (grey circles) and Ure2p1-93 (solid triangles)
monitored by thiolavin T binding and fluorescence. B, time course analysis by SDS PAGE of the generation of Ure2p1-93 by cleavage of the
variant Ure2pI91EGR94 by Factor Xa. The gel was silver-stained, the molecular weight markers are shown on the left and the position of
Ure2p1-93 is indicated by the arrow. C and D, Electron micrographs of negatively stained samples of Ure2pI91EGR94 and Ure2p1-93, re-
spectively. The bar represents 0.1 m.
Ure2p1-93 is barely assembling as demonstrated by the ex-
tablishes under non-reducing conditions. This strategy re-
tent of thioflavin T binding. This suggests that the assembly
vealed that amino acid sequences centered around residue 6
of Ure2p1-93 is limited either by its release from full-length
in the N-terminal domain of Ure2p and residue 137 in the C-
Ure2p after cleavage of the peptide bond linking Ure2p1-93
terminal moiety interact through intramolecular interactions
to the rest of the protein or by the acquisition of an assembly
both in soluble and fibrillar Ure2p [59] consistent with the
competent conformation or both.
finding that Ure2p94-354 is tightly involved in the fibrillar
scaffold [41]. Finally, the analysis of the changes in Ure2p
STRUCTURE OF Ure2p WITHIN THE FIBRILLAR
structure associated with its assembly into fibrils by solvent
FORM OF THE PROTEIN
accessibility studies using hydrogen/deuterium exchange
monitored by mass spectrometry revealed that Ure2p under-

To document the structure of the flexible N-terminal do-
goes limited structural changes upon assembly [60]. The
main of Ure2p, critical for prion propagation, and determine
only peptide shown to exhibit a protection against the sol-
whether Ure2p1-93 interacts with the C-terminal domain of
vent of the same order of magnitude as that of amino acid
the protein within the fibrils, Ure2p variants with cysteine
stretches of significant length involved in the systematically
residues in the N- and C-terminal domains were generated.
H-bonded -sheet core of amyloid forming peptides such as
Such variants allow determining whether the N- and C-
PrP [61], A [62] and HET-s [63] fibrils was a four amino
terminal regions interact in soluble and fibrillar Ure2p. In-
acid stretch spanning residues 13-16 [60].
deed, if a cysteine residue from the N-terminal domain lies
within soluble or fibrillar Ure2p at 3-4 Å from another cys-

To further describe the potential interactions between the
teine residue in the C-terminal domain a disulfide bond es-
polypeptide chains within the fibrillar forms of the Q- and N-

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256 Current Alzheimer Research, 2008, Vol. 5, No. 3
Bousset et al.
rich yeast prions, it is important to review the experimental
their fundamental ability to mediate efficiently protein-
and theoretical structures that are either available or that
protein interactions that may lead to assembly into macromo-
have been proposed for Q- and N-rich motifs involved in
lecular structures.
protein-protein interactions.

Earlier studies and calculations from Max Perutz and
coworkers led them to propose a cylindrical structure for Q-
EXPERIMENTAL AND THEORETICAL STRUC-
rich polypeptides [67] (Fig. 5B, top). Within this structure
TURES OF Q- AND N-RICH MOTIFS INVOLVED IN
and in contrast with the polar zipper structure described
PROTEIN-PROTEIN INTERACTIONS
above, the lateral chains of Q residues are exposed alterna-

Q- and N-rich sequences exist in a wide range of pro-
tively toward the exterior and the interior of the cylinder and
teins. Some of these polypeptides assemble into fibrils while
interact with water molecules. Residues from neighboring
others are unable to do so. Two structures of overlapping Q-
turns are hydrogen bonded by both main chain and side
and N-rich peptides reproducing a fragment of the prion
chain amides with every carboxyl (C=O) in one turn bonded
Sup35p and one structure of a peptide reproducing an amino
to an amide (N-H) in the next turn (Fig. 5B, bottom). This
acid stretch of human prion protein are available at an atomic
structure contains a dry polar zipper made of the backbone
resolution [64-65]. These peptides: GNNQQNY, NNQQNY
C=O--H-N hydrogen bonds and differs fundamentally from
from Sup35p and SNQNNF from PrP form fibrils and micro-
the structure where the lateral chains interdigitate to form a
crystals revealing their atomic structures. In the crystals
dry polar zipper in that the lateral chains are exposed to the
these peptides are extended, parallel and in register. They
solvent and have the potential to establish lateral protein-
appear hydrogen bonded in a standard parallel, in register -
protein interactions. Such lateral interaction may account for
sheet. Within each pair of sheets, the strands in one sheet are
an important structural feature of fibrils made of Q-rich
antiparallel to those in the facing sheet. In addition, each
polypeptides as they appear to be made of bundled protofi-
sheet is shifted along the axis relative to its partner by half of
brils. Indeed, it is hypothesized that several cylindrical struc-
the strand-strand separation. The side chains extending from
tures described above interact laterally to form the mature
one sheet interdigitate with those extending from the facing
fibril.
sheet like the teeth of a zipper and form van der Waals inter-
actions and water molecules are excluded from the interface
CONCLUSION
(Fig. 5). In the case of GNNQQNY, each peptide forms 11

While there is general agreement on the role of Q and N
hydrogen bonds with its two neighbours within the same
residues in mediating protein-protein interactions, three dif-
sheet four of which are amide-amide hydrogen bonds be-
ferent structures have been used so far to describe at the
tween pairs of identical N or Q residues in adjacent mole-
atomic level the interactions these residues are capable of
cules and five of which are backbone C=O--H-N hydrogen
establishing to drive, in particular the assembly of Q- and N-
bonds. The remaining hydrogen bonds come from side
rich polypeptides into protein fibrils. Two of these models
chains (Fig. 5A, top).
describe the formation of dry zippers [64-65, 67]. However,

The packing of peptide SNQNNF from PrP within the
while in the first structure [64-65], it is the side chains of Q
crystals differs from that of GNNQQNY from Sup35p in that
and N residues extending from one sheet that interdigitate
instead of having the side chains facing each other and the
with those extending from the facing sheet thus forming a
two constituent sheets edges facing up, the side chains are
zipper-like interface, the second model [67] proposes that the
packed face to back with the two constituent sheets edges
main chains of Q residues and by analogy N residues are the
facing up (Fig. 5A, bottom).
driving force of formation of a zipper-like interface with the
amino acid side chains exposed to water. The third model

A crystal-structure of a larger Q-rich polypeptide (the N-
[66] suggests that Q and N residues facilitate protein-protein
terminal domain of histone deacetylase 4) was made avail-
interactions the strength of which depends on the number of
able recently [66]. Within this structure, Q residues are in an
i- hydrogen bonds established between their amide groups
all -helical instead of a -rich structure (Fig. 5C, top). The
and positively or negatively charged amino acid residues and
atomic structure reveals that while Q residues are random
ii- van der Waals contacts through their aliphatic side chains.
within the primary structure, they appear exposed to the sol-
vent and involved in a large number of hydrogen bonds

The first model [64-65] is derived from very short pep-
through their amide group. Indeed, some of these Q residues
tides nearly exclusively made of Q and N residues. The sec-
stabilize the helical structure of the protein, while others are
ond model [67] is derived from polypeptides virtually unlim-
involved in interhelical interactions (Fig. 5C, bottom). Fi-
ited in length but made exclusively of Q residues. These two
nally, a number of Q side chains form van der Waals con-
models therefore neither take into account the contribution
tacts, thus, stabilizing a tetrameric form of the protein.
within Q and N-rich sequences of the 18 other amino acid
residues nor the role of residues that play the role of gate

These structural data strongly suggest that by establishing
keepers and disfavour aggregation [68]. The third model [66]
i- hydrogen bonds between their amide groups and positively
corresponds to situations encountered in a biological context
or negatively charged amino acid residues and ii- van der
where polyQ and by analogy polyN stretches are interrupted
Waals contacts through their long aliphatic side chains, Q
by other amino acid residues. Under such physiological con-
and N residues facilitate protein-protein interactions that
ditions, Q and N residues establish hydrogen bonds with
may lead depending on the number of established bonds to
positively and negatively charged residues and van der
reversible or irreversible polypeptide assemblies. Further-
Waals contacts through their aliphatic side chains. The num-
more, these studies clearly indicate that the arrangement of Q
ber of established bonds is what defines the reversibility of
and N residues in -rich structures is not a prerequisite for
such interactions.

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Assembly of the Asparagine- and Glutamine-Rich Yeast Prions
Current Alzheimer Research, 2008, Vol. 5, No. 3 257



















Fig. (5). Structures of Q- and N-rich motifs involved in protein-protein interactions. A, microcrystal structures of the peptides GNNQQNY
from Sup35p (top) and SNQNNF from PrP (bottom) [Ref. 64-65]. In the two cases, three stacked peptides face a single peptide. B, cylindrical
structure for Q42 polypeptides (top). The lateral chains of Q residues are exposed alternatively toward the exterior and the interior of the cyl-
inder (bottom, left). Residues from neighboring turns are hydrogen bonded by both main chain and side chain amides with every carboxyl
(C=O) in one turn bonded to an amide (N-H) in the next turn (bottom, right). The lateral chains are exposed to the solvent and have the poten-
tial to establish lateral protein-protein interactions [Ref. 67]. C, structure of larger Q-rich polypeptide (the N-terminal domain of histone
deacetylase 4). Q residues are random within the primary structure, they appear however exposed to the solvent. They stabilize the helical
structure of the protein and its tetrameric form (top) through hydrogen bonds and van der Waals contacts (bottom) [Ref. 66]. The structures
were generated with PyMol [http://pymol.sourceforge.net/]. The PDB coordinates used for the peptides GNNQQNY and SNQNNF are 1YJP
and 2OL9, respectively, and that of the N-terminal domain of histone deacetylase 4 is 2H8N. Oxygen and nitrogen atoms are colored red and
blue, respectively, throughout the figure. The peptide backbones have different colors. The hydrogen bonds are represented by dashed lines.
In the case of the prion Ure2p where a hydro-
in the case of the leucine zipper motif and ii- lead to thera-
gen/deuterium exchange mass spectrometric analysis of the
peutic applications.
conformational changes accompanying assembly into fibrils
has been carried out, neither the side nor the main chains of
ACKNOWLEDGEMENTS
the N- and Q-rich domain are involved in a dry interface

L.B. and R.M. are supported by the French Ministry of
where hydrogen exchange is not permitted. In contrast, the
Eduction, Research and Technology through the Grouppe-
data are fully consistent with the model where these residues
ment d’intérêts Scientifiques Prion, the CNRS and the ANR.
mediate protein-protein interactions via hydrogen bonding
and van der Waals contacts. In addition, and in agreement
ABBREVIATIONS
with the view that amino acid residues located within polyN
and Q stretches, in other words the natural context of these
Q =
Glutamine
stretches, have a critical effect on their propensity to estab-
N =
Asparagine
lish protein-protein interactions, the insertion of amino acid
residues other than N and Q affect significantly the assembly
G =
Glycine
of Ure2p into fibrils [69].
S =
Serine

Further biochemical studies and high resolution struc-
T =
Threonine
tures are needed to define a potential Q and N-rich consensus
PDB = Protein data bank
motif that drives protein-protein interactions leading, in par-
ticular, to the formation of fibrillar structures. Such efforts
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