On the aggregation properties of FMRP – a link with the
FXTAS syndrome?
Ljiljana Sjekloc
´
a*, Kris Pauwels and Annalisa Pastore
MRC National Institute for Medical Research, London, UK
Introduction
The fragile X mental retardation protein (FMRP) is
an  70 kDa human protein encoded by the X-linked
gene FMR1 which is expressed in different organs,
most prominently in brain and gonads [1–3]. FMRP is
a multi-domain protein which contains two Tudor
domains connected to two protein K homology (KH)
domains by an ca. 80 amino acids residue long linker.
This region is followed by a putative intrinsically
unstructured region which contains also arginine- and
glycine-rich (RGG) motifs [4,5]. Co-presence of differ-
ent nucleic acid binding domains in FMRP suggests
that the protein has a prominent capacity to bind
nucleic acids, in particular RNA, as experimentally
confirmed both in vitro and in vivo [6,7].
The cellular role of FMRP is not well understood.
Experimental evidence shows that FMRP binds co-
transcriptionally to certain messenger RNAs forming
messenger ribonucleoprotein (mRNP) particles, which
are exported from the nucleus to the cytoplasm [8]. In
the cytoplasm FMRP associates to microtubules, to
polysomes and to mRNPs and permits the mRNP par-
ticles to be delivered to distal dendrite sites [9]. It has
Keywords
aggregation; fragile X mental retardation

Structured digital abstract
l
FXR2P binds to FXR2P by fluorescence technology (View interaction)
l
FMRP binds to FMRP by electron microscopy (View interaction)
l
FXR1P binds to FXR1P by electron microscopy (View interaction)
Abbreviations
CD, circular dichroism; FMRP, fragile X mental retardation protein; FXR, fragile X related; FXS, fragile X syndrome; FXTAS, fragile X
associated tremor ataxia syndrome; KH, K homology; mRNP, messenger ribonucleoprotein; NDF, N-terminal domain; NES, nuclear export
signal; rCGG, cytosine guanine triribonucleotide; ThT, thioflavin T.
1912 FEBS Journal 278 (2011) 1912–1921 ª 2011 The Authors Journal compilation ª 2011 FEBS
also been shown that messenger RNAs bound to
FMRP are translationally repressed and that, in neu-
rons, FMRP acts in an activity-dependent manner as
an inhibitor of translation initiation ([10] and refer-
ences therein).
Most studies on FMRP are related to its functions
in brain neurons for two reasons. First, the lack of
functional FMRP, due to transcriptional silencing of
FMR1 gene, causes a neurodevelopmental disorder,
fragile X mental retardation syndrome (FXS), the most
common inherited mental disorder in humans. FXS is
characterized by mild to severe mental retardation,
autistic behaviour and, in male patients, macro-orchi-
dism [11]. Second, alteration of FMRP expression,
characterized by increased levels of FMR1 mRNA and
decreased protein levels, can lead to a late onset neuro-
degenerative disorder, the fragile X associated tremor
ataxia syndrome (FXTAS), with symptoms similar to

embryonic Fmr1 KO STEK cells induce formation of
cytoplasmic stress granules in which mRNAs are
trapped into repressed mRNP granules [24,25]. In a
recent study, we investigated the oligomerization
properties of human FXR proteins and showed that,
in vitro, multi-domain constructs from the highly con-
served N-terminus have an elevated tendency to aggre-
gate [26]. They self-assemble not just by forming
dimers but through a more complex pattern of self-
association which proceeds in a continuous way from
the monomer to large molecular weight aggregates via
formation of dimeric species. We proposed that this
behaviour is typical of ‘complex-orphan proteins’, i.e.
proteins which exist in the cell as part of large molecu-
lar assemblies. When produced in isolation, they have
an elevated tendency to self-associate.
To further characterize the nature of aggregation of
FXR proteins, we have carried out a study of their
aggregation properties using different approaches. We
identified by in silico analysis potential hot-spots of
aggregation ⁄ fibrillation and showed that they all
cluster in the protein N-terminus. We then studied the
aggregation behaviour of various constructs from
FMRP, FXR1P and FXR2P using complementary
biophysical techniques. We demonstrate that not only
do all constructs have an intrinsic tendency to aggre-
gate but they also undergo an irreversible conforma-
tional transition towards b-enriched structures which
are typical of amyloidogenic diseases such as Alzhei-
mer’s, Parkinson’s and Huntington’s diseases. The

presence of several potential aggregation foci all
grouped in the highly conserved N-terminus.
Temperature induces a conformational transition
of FMRP domains
We tested experimentally the role of the different
regions in aggregation ⁄ fibrillation using single-and
multiple-domain constructs from human FXR proteins
which we knew from previous extensive characteriza-
tion are stable to degradation [26] (Fig. 2). Although
monodispersed at sufficiently low concentrations, we
had previously demonstrated that they all have a
strong tendency to aggregate [26]. To check whether
aggregation is associated with misfolding, we probed
their secondary structure as a function of temperature
by far-UV circular dichroism (CD). We first analysed
the secondary structure content at different tempera-
tures of FMRP Nt-KH1 (residues 1–280), the longest
of the FMRP fragments we could obtain in a stable
form (Fig. 3A). The spectrum of Nt-KH1 is typical of
an a–b fold at 20 °C, as expected from the presence in
the construct of Tudor and KH domains [4,28]. At
higher temperatures (40–45 °C), the conformation
starts to change (Fig. 3B). At 55 °C, the spectrum
becomes typical of an all-b protein indicating a
profound structural rearrangement with a minimum
around 215 nm. The transition is irreversible, as
the CD spectra of samples treated at 45 °C remain
Fig. 1. Sequence alignment of FXR proteins and indication of fibrillogenic regions. The alignment was produced and colour coded according
to
CLUSTALW2 [27]. Extra rows were added below for the rulers relative to human FMRP, FXR1P and FXR2P. The regions predicted as fibrillo-

(Fig. 3E). For comparison, the two longer constructs
Nt and Nt-KH1 incubated at 45 °C did not reach,
over the same time, the minimum CD signal (Fig. 3E)
observed for the corresponding samples at 55 °C. A
similar experiment was performed at 50 °C and
resulted in a faster conformational transition compared
with 45 °C (Fig. 3F). At this temperature, the intensi-
ties of the NDF and Nt-KH1 spectra reached a maxi-
mum after 40 and 120 min, respectively. Nt underwent
a conformation transition at 50 °C which was not,
however, complete during the time course of the exper-
iment (3 h). This suggests that, under the same experi-
mental conditions, the region C-terminal to the NDF,
comprising the linker between NDF and KH1, has a
prominent role in aggregation.
Taken together, these data show that different
domains of the conserved region of FXR proteins rear-
range their structure upon temperature treatment. In
all cases examined this rearrangement occurs with very
similar modalities and results in a significant enrich-
ment of the b content.
Tendency to misfold is a conserved feature of
FXR proteins
To extend our studies to other FXR proteins, we used
the human FMRP paralogues FXR1P and FXR2P.
The secondary structure of FXR1P Nt-NES was first
Fig. 3. Spectroscopic study of temperature-induced conformational changes of FMRP. (A) Far-UV CD spectra of Nt-KH1 of FMRP recorded
at 20 °C (black line) or 55 °C (dotted line) and expressed in molar ellipticity of Nt-KH1. (B) Temperature course at 220 nm, in molar ellipticity,
of FMRP NDF (curve a), Nt (curve b) and Nt-KH1 (curve c). The rate of temperature increase was 1 °CÆmin
)1

Since the duration of temperature treatment plays a
role in the observed process, we tested if prolonged
incubation could lead to a-to-b conformational transi-
tion also at 37 °C, i.e. close to the physiological tem-
perature at which FMRP functions in human cells.
Initially, incubation of FMRP Nt-KH1 (5 lm)at
37 °C did not cause a significant secondary structure
perturbation, but a conformational transition to a
b-enriched structure was observed after a 45-h incuba-
tion (Fig. 5A). The lag time decreased to 16 h at con-
centrations threefold or sixfold higher (15 and 30 lm
respectively), as expected for a concentration-depen-
dent phenomenon such as aggregation (Fig. 5B,C).
The final intensity of the recorded CD signal is very
similar to that recorded at 55 °C, suggesting not
only that a similar process takes place at both temper-
atures but also that the final states are structurally
comparable.
Freshly prepared FMRP Nt-KH1 samples (30 lm)
are monomeric and monodispersed, and if stored at
4 °C they remain mainly monomeric with a small but
detectable increase of dimeric species as time pro-
ceeds, i.e. after 16 h incubation. The size exclusion
chromatograms of these samples incubated at 37 °C
over the same time show the appearance of high
molecular weight species which are absent both in
fresh samples and in samples incubated at 4 °C
(Fig. 5D). We can conclude that recombinant Nt-
KH1 of FMRP has an intrinsic tendency to aggregate
in vitro also at physiological temperature in native-like

not show further increase and a net decrease was
observed after 96 h, probably caused by fibre sedimen-
tation (data not shown).
To verify the morphology of the end-states of aggre-
gation, we used transmission electron microscopy and
examined samples after the conformational transition
Fig. 5. Following the conformational transi-
tion of FMRP Nt-KH1 at 37 °C and different
incubation times as a function of protein
concentration. (A), (B), (C) Comparison of
the FMRP Nt-KH1 CD spectra before (con-
tinuous line) and after (dotted line) incuba-
tion at 37 °C using 5, 15 and 30 l
M protein
concentrations, respectively. (D) Size exclu-
sion chromatography elution profile of
FMRP Nt-KH1: the continuous line chro-
matogram derives from freshly prepared
Nt-KH1, the broken line chromatogram is
the profile of the same sample kept at 4 °C
for 16 h, and the dotted line chromatogram
corresponds to a sample incubated at 37 °C
for 16 h.
A
B
C
D
Fig. 6. Testing the fibrillogenic properties of
FXR proteins. (A) ThT fluorescence assay on
FXR2P Nt-NES treated over the temperature

(7 nm) and variable lengths that very rarely exceeded
100 nm (Fig. 6B). Interestingly, we also observed dense
networks of long linear and unbranched fibrils with a
10-nm diameter, which displayed repeating segments
and twists. The FMRP Nt-KH1 samples contained
globular particles with an average diameter of 24 nm,
often decorated with stain, as well as clustered deposits
of fibrils with an average diameter of 6 nm (Fig. 6C).
FX1RP Nt-NES aggregates have a curved appearance,
with an apparent average diameter of 10 nm (Fig. 6D).
They also clustered together and were often found to
be decorated with the uranyl acetate stain. Taken
together these results confirm a marked tendency of
FXR constructs to fibrillation.
Discussion
We have shown here that different fragments of FXR
proteins not only have a strong tendency to aggregate
as previously described [26] but also undergo an irre-
versible conformational transition which leads to a sig-
nificant increase in their b-structure content. Several
conserved putative aggregation and amyloidogenic
hot-spots were predicted by in silico analysis of the
FXR amino acid sequences. They are all grouped in
the highly conserved (more than 70–80% identity and
80–90% similarity) N-terminal half of the proteins
which is also the region involved in most of the inter-
actions with the FXR cellular partners [30], suggesting
that the aggregation hot-spots could have a prominent
role in determining the hetero- and self-assembly
behaviours of the full-length proteins. By combining

ditions. This is the case for instance of the globular Jo-
sephin domain of ataxin-3, the protein responsible for
the misfolding Machado–Joseph disease: we have
recently shown that Josephin aggregation and misfold-
ing is promoted by exposed hydrophobic patches
involved in recognition of its natural partner ubiquitin,
thus suggesting a link between normal function and
misfolding [31]. Likewise, the globular domain of the
prion protein contains a seeding region, H2H3, which
retains its fold during the early stages of unfolding [32].
It has been suggested that in many proteins related to
conformational diseases aggregation ⁄ amyloidogenic
regions coincide with interaction surfaces [33–35].
Our results bear a number of interesting conse-
quences. First, the strong tendency to aggregate of
FXR proteins could help us to understand the driving
forces that lead to granular formations and eventually
understand more about their functional role. The find-
ings presented in this study also suggest interesting
possibilities for the ability of this family of proteins to
contribute to both early life syndromes such as FXS
(for instance through destabilizing mutations) and
aggregation-related neurodegeneration later in life;
such could be the case of FXTAS. The latter is a par-
ticularly interesting possibility since it could shed new
light onto a still poorly understood syndrome:
although RNA aggregation is thought to be an impor-
tant driving force for formation of the pathological
neuronal intranuclear RNP inclusions observed in
FXTAS patients, little is known about the factors

(Q5F3S6), frog (P51113, Q6GLC9, P51115), zebra fish
(Q7SYM7, Q7SXA0, Q6NY99) and fruit fly (Q9NFU0)
FXR proteins were aligned using program clustalw2 [27].
These sequences were also searched for putative determi-
nants of aggregation and amyloidogenesis by the following
consensus prediction tools: aggrescan (http://bioinf.
uab.es/aggrescan/) for prediction of hot-spots for aggrega-
tion in polypeptides; pasta ( />pasta/) for prediction of amyloid-like structure aggregation;
amylpred ( to
predict features related to the formation of amyloid fibrils;
tango ( for prediction of
sequence-dependent and mutational effects on the aggrega-
tion of the peptides and proteins; and waltz (http://
waltz.switchlab.org/) for predicting amyloidogenic regions
in protein sequences.
Cloning, protein expression and purification
The constructs studied in this paper were produced accord-
ing to procedures previously described [26]. In short, clones
of human FMR1, FXR1 and FXR2 were used as templates
for DNA amplification by PCR. PCR amplicons encoding
different fragments of the conserved region of FXR pro-
teins were cloned into a modified pET-24 vector (Novagen,
Gibbstown, NJ, USA) encoding an amino terminal Trx
(thioredoxin)-His6-tag and a tobacco etch virus (TEV) pro-
tease cleavage site.
Escherichia coli BL21 STAR (DE3) cells transformed
with plasmids encoding different FXR fragments were
grown at 37 °C in Luria–Bertani medium containing appro-
priate antibiotic. Protein over-expression was induced with
0.2 mm isopropyl thio-b-d-galactoside after the cell culture

Aggregation studies by CD and size exclusion
chromatography
CD spectra were recorded using a Jasco J-715 spectropola-
rimeter equipped with a thermostatted cell holder controlled
by a Jasco Peltier element, at different temperatures, over a
wavelength range from 260 to 190 nm in quartz cuvettes
(Hellma) of path length appropriate to protein concentra-
tion of the samples, i.e. 1 mm for 5 lm (0.15 mgÆmL
)1
),
0.2 mm for 15 lm (0.5 mg ÆmL
)1
) and 0.1 mm for 30 lm
(1 mgÆmL
)1
). Thermally induced denaturation transitions
were monitored by CD absorption at 220 nm from 10 to
95 °C, in 1-°C steps and with an equilibration time of
1 minÆ°C
)1
. Reversibility was tested by performing an
inverse temperature scan. The purified recombinant proteins
were in 20 mm Tris ⁄ HCl (pH 8.0), 1 mm b-mercaptoetha-
nol. To monitor progression of protein aggregation over
time, protein samples were incubated at 37 °C and CD
spectra were recorded at different time points (1, 6, 16, 24,
45, 72 h, 1 week).
Analytical size exclusion chromatography was carried out
by injecting 100 lL of samples (30 lm) into a Superdex 200
10 ⁄ 300 GL column.

developer (Ilford) and Hypam fixer (Ilford) for 5 min each.
Acknowledgements
We thank Steve Martin for help with CD and fluores-
cence studies, Lesley Calder for support with electron
microscopy analysis and Steve Howell for mass spec-
trometry analysis. We are grateful to Cesira de Chiara
and Laura Masino for critical discussion and assis-
tance in graphic elaboration of CD results. We
acknowledge support from the MRC (Grant ref.
U117584256). Kris Pauwels is the recipient of an
EMBO long-term postdoctoral fellowship (ALTF 512-
2008).
References
1 Bakker CE, de Diego Otero Y, Bontekoe C, Raghoe P,
Luteijn T, Hoogeveen AT, Oostra BA & Willemsen R
(2000) Immunocytochemical and biochemical character-
ization of FMRP, FXR1P, and FXR2P in the mouse.
Exp Cell Res 258, 162–170.
2 Devys D, Lutz Y, Rouyer N, Bellocq JP & Mandel JL
(1993) The FMR-1 protein is cytoplasmic, most abun-
dant in neurons and appears normal in carriers of a
fragile X permutation. Nat Genet 4, 335–340.
3 Tamanini F, Willemsen R, van Unen L, Bontekoe C,
Galjaard H, Oostra BA & Hoogeveen AT (1997) Differ-
ential expression of FMR1, FXR1 and FXR2 proteins
in human brain and testis. Hum Mol Genet 6, 1315–
1322.
4 Ramos A, Hollingworth D, Adinolfi S, Castets M,
Kelly G, Frenkiel TA, Bardoni B & Pastore A (2006)
The structure of the N-terminal domain of the fragile X

12 Brouwer JR, Willemsen R & Oostra BA (2009) The
FMR1 gene and fragile X-associated tremor ⁄ ataxia syn-
drome. Am J Med Genet B Neuropsychiatr Genet 150B,
782–798.
13 Oostra BA & Willemsen R (2009) FMR1: a gene with
three faces. Biochim Biophys Acta 1790, 467–477.
14 Guduric-Fuchs J, Mohrlen F, Frohme M & Frank U
(2004) A fragile X mental retardation-like gene in a
cnidarian. Gene 343, 231–238.
15 Anderson P & Kedersha N (2006) RNA granules. J Cell
Biol 172, 803–808.
16 Christie SB, Akins MR, Schwob JE & Fallon JR (2009)
The FXG: a presynaptic fragile X granule expressed in
a subset of developing brain circuits. J Neurosci 29,
1514–1524.
17 Huot ME, Bisson N, Davidovic L, Mazroui R, Labelle
Y, Moss T & Khandjian EW (2005) The RNA-binding
protein fragile X-related 1 regulates somite formation in
Xenopus laevis. Mol Biol Cell 16
, 4350–4361.
Aggregation properties of fragile X related proteins L. Sjekloc
´
a et al.
1920 FEBS Journal 278 (2011) 1912–1921 ª 2011 The Authors Journal compilation ª 2011 FEBS
18 Moser JJ & Fritzler MJ (2010) Cytoplasmic ribonucleo-
protein (RNP) bodies and their relationship to GW ⁄ P
bodies. Int J Biochem Cell Biol 42, 828–843.
19 Tassone F, Iwahashi C & Hagerman PJ (2004) FMR1
RNA within the intranuclear inclusions of fragile X-
associated tremor ⁄ ataxia syndrome (FXTAS). RNA Biol

26 Sjekloca L, Konarev PV, Eccleston J, Taylor IA,
Svergun DI & Pastore A (2009) A study of the ultra-
structure of fragile-X-related proteins. Biochem J 419,
347–357.
27 Larkin MA, Blackshields G, Brown NP, Chenna R,
McGettigan PA, McWilliam H, Valentin F, Wallace
IM, Wilm A, Lopez R et al. (2007) Clustal W and
Clustal X version 2.0. Bioinformatics 23, 2947–2948.
28 Musco G, Stier G, Joseph C, Castiglione Morelli MA,
Nilges M, Gibson TJ & Pastore A (1996) Three-dimen-
sional structure and stability of the KH domain:
molecular insights into the fragile X syndrome. Cell 85,
237–245.
29 LeVine H III (1993) Thioflavine T interaction with syn-
thetic Alzheimer’s disease beta-amyloid peptides: detec-
tion of amyloid aggregation in solution. Protein Sci 2,
404–410.
30 Bardoni B, Davidovic L, Bensaid M & Khandjian EW
(2006) The fragile X syndrome: exploring its molecular
basis and seeking a treatment. Expert Rev Mol Med 8,
1–16.
31 Masino L, Nicastro G, Calder L, Vendruscolo M &
Pastore A (2010) Functional interactions as a survival
strategy against abnormal aggregation. FASEB J 25,
45–54.
32 Adrover M, Pauwels K, Prigent S, de Chiara C, Xu Z,
Chapuis C, Pastore A & Rezaei H (2010) Prion fibrilliza-
tion is mediated by a native structural element that com-
prises helices H2 and H3. J Biol Chem 285 , 21004–21012.
33 Castillo V & Ventura S (2009) Amyloidogenic regions

updates and new developments. Nucleic Acids Res 37,
D229–D232.
L. Sjekloc
´
a et al. Aggregation properties of fragile X related proteins
FEBS Journal 278 (2011) 1912–1921 ª 2011 The Authors Journal compilation ª 2011 FEBS 1921