Eur. J. Biochem.

271, 31553170 (2004)
FEBS 2004

doi:10.1111/j.1432-1033.2004.04245.x

Structural and functional analysis of ataxin-2 and ataxin-3

Mario Albrecht1,*, Michael Golatta2,*, Ullrich Wullner3 and Thomas Lengauer1
1

Max-Planck-Institute for Informatics, Saarbrucken, Germany; 2Institute for Medical Biometry, Informatics, and Epidemiology,
University of Bonn, Germany; 3Department of Neurology, University of Bonn, Germany

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are

autosomal-dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding
regions of the genes encoding ataxin-2 and ataxin-3,
respectively. To provide a rationale for further functional
experiments, we explored the protein architectures of ataxin2 and ataxin-3. Using structure-based multiple sequence
alignments of homologous proteins, we investigated
domains, sequence motifs, and interaction partners. Our
analyses focused on presumably functional amino acids and

the construction of tertiary structure models of the RNAbinding Lsm domain of ataxin-2 and the deubiquitinating
Josephin domain of ataxin-3. We also speculate about distant evolutionary relationships of ubiquitin-binding UIM,
GAT, UBA and CUE domains and helical ANTH and
UBX domain extensions.

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are

autosomal-dominantly inherited, neurodegenerative disorders [1,2]. SCA3 has also been known as Machado
Joseph disease (MJD), and SCA2 and SCA3 belong to a
heterogeneous group of trinucleotide repeat disorders. This
group includes Huntington disease (HD), dentatorubralpallidoluysian atrophy (DRPLA), and other spinocerebellar
ataxia types such as SCA1, SCA7 and SCA17 [37]. The age
of onset of SCA2 and SCA3 is in the third to fourth decade
[8]. The disorders share common phenotypic features such
as the degeneration of specic vulnerable neuron populations and the presence of intracellular aggregations of the
mutant proteins in affected neurons. In contrast, the
expression of the disease-associated genes occurs in a great
variety of tissues and is not restricted to neuronal cells.
The SCA2 and SCA3/MJD genes have been mapped to
chromosomes 12q24.1 and 14q32.1 [1,2]. The common
underlying genetic basis of SCA2 and SCA3 is the
expansion of a CAG repeat region beyond a certain threshold. These CAG repeats encode a polyglutamine (polyQ)
tract in the respective proteins ataxin-2 and ataxin-3. The
polyQ stretch in ataxin-2 lies near the N-terminus at the 5end of the coding region of exon 1 [9], but the polyQ region

of ataxin-3 is contained in exon 10 close to the C-terminus

[10]. While ataxin-2 is located predominantly in the Golgi
apparatus [11], ataxin-3 is found in both the nucleus and the
cytoplasm of cells [12].
To provide a rationale for further experiments, we
characterized the protein architectures of ataxin-2 and
ataxin-3 and investigated domains, sequence motifs, and
interaction partners. To explore the functional implications,
we assembled a multiple sequence alignment for the Lsm
domain of ataxin-2 homologues including the yeast
homologue Pbp1. We also constructed a 3D structural
model for the RNA-binding Lsm domain of ataxin-2.
Similarly, we used a structure-based multiple sequence
alignment of the Josephin domain of ataxin-3 homologues
to derive a 3D model of this domain and to analyse specic
residues involved in deubiquitination.

Correspondence to M. Albrecht, Max-Planck-Institute for Informatics,

Stuhlsatzenhausweg 85, 66123 Saarbrucken, Germany.
E-mail: mario.albrecht@mpi-sb.mpg.de
Abbreviations: A2BP1, ataxin-2 binding protein 1; DRPLA, dentatorubral pallidoluysian atrophy; DUB, deubiquitinating enzymes;
HD, Huntington disease; MJD, MachadoJoseph disease; NLS,
nuclear localization signal; OTU, otubains; PABP, poly(A)-binding
protein; RMSD, root mean square deviation; SCA, spinocerebellar
ataxia; SnRNPs, small nuclear ribonucleoproteins; UBP, ubiquitinspecic protease; UCH, ubiquitin C-terminal hydrolase; UIM,
ubiquitin-interacting motif; VCP, valosin-containing protein.
*Note: M. Albrecht and M. Golatta contributed equally to this work.
(Received 6 April 2004, accepted 7 June 2004)

Keywords: spinocerebellar ataxia; MachadoJoseph disease; polyglutamine disorder; ubiquitin; valosin-containing

protein.

Materials and methods

Protein sequences were retrieved from the NCBI [13],
Ensembl [14], and SWISS-PROT/TrEMBL (SPTrEMBL)
[15] databases and protein domain architectures from the
Pfam [16] and SCOP [17] databases. Sequence accession
numbers are given in the respective gure legends and
Tables S1 and S2. Species names are abbreviated by rst
letters (Table S3). Protein structures were obtained from the
PDB database [18]. The secondary structure assignments of
PDB structures were taken from the DSSP database [19]. A
single capital letter appended to the actual PDB identier
denotes the chosen structure chain. We used the PSI-BLAST
suite of programs [20] to search for homologues (E-value
cut-off 0.005) and the web servers PSIPRED [21], SAM-T99
[22], and SSpro2 [23] to predict the secondary structure of
proteins and to form a consensus prediction by majority
voting [24]. To predict intrinsically unstructured and
disordered regions in proteins, we explored the consensus
of the results returned by the DisEMBL [25], DISOPRED
[26], GlobPlot [27], NORSp [28] and PONDR [10] online

3156 M. Albrecht et al. (Eur. J. Biochem. 271)

servers. The nuclear localization signals in ataxin-3 homologues were discovered with help of the prediction server
PSORT II [29].
Multiple sequence alignments were assembled by means
of T-COFFEE [30] and improved manually by minor
adjustments based on structure prediction results and pairwise structure superpositions computed by the program CE
[31]. The root mean square deviations (RMSDs) were taken
from the CE superpositions. We investigated the results of all
state-of-the-art fold recognition methods available via the
online meta-server BioInfo.PL [32], which contacts a dozen
other state-of-the-art prediction servers (the names of which
are listed on the web site http://Bioinfo.PL/Meta/). The
associated 3D-Jury system allows for the comparison and
evaluation of the predicted 3D models in a consensus view
[33]. To model the protein structure of ataxin-2 and ataxin3, we submitted the constructed sequencestructure alignments to the 3D modelling server WHAT IF [34]. The
sequence alignments depicted in the gures were prepared in
the SEAVIEW editor [35] and illustrated by the web service
ESPript [36]. The protein structure images were drawn
in the Accelrys Discovery Studio ViewerLight. The online
version of this manuscript contains supplementary material,
and our web site will provide additional pictures.

Results and discussion

Protein architecture of ataxin-2
Ataxin-2 has 1312 residues (including 22 glutamines of the
polyQ stretch) and a molecular mass of  140 kDa. Ataxin2 is a highly basic protein except for one acidic region
(amino acid 254475) containing 46 acidic amino acids
(Fig. 1). This region covers roughly exons 27 and is
predicted to consist of two globular domains named Lsm
(Like Sm, amino acid 254345) [37] and LsmAD (Lsmassociated domain, amino acid 353475). The LsmAD

FEBS 2004

domain of ataxin-2 contains both a clathrin-mediated

trans-Golgi signal (YDS, amino acid 414416) and an
endoplasmic reticulum (ER) exit signal (ERD, amino acid
426428) [11,38]. It is composed mainly of a-helices
according to the results from secondary structure prediction
servers.
The rest of ataxin-2 outside of the Lsm and LsmAD
domains is only weakly conserved in eukaryotic ataxin-2
homologues and is predicted to be intrinsically unstructured
according to the consensus result from the DisEMBL,
DISOPRED, GlobPlot, NORSp and PONDR online
servers. These nonglobular, exible N- and C-terminal tails
(amino acid 1253 and 4761312) contain the polyQ region
(amino acid 166187), several highly conserved short
sequence motifs as possible protein interaction sites, and
conspicuous (R)RG peptides at the C-terminus of the
LsmAD domain. One of the sequence motifs constitutes a
putative PABP [poly(A)-binding protein] interacting motif
PAM2 (amino acid 908925) [39], and (R)RG peptides are
well-known to bind RNA in other proteins [40]. The N- and
C-terminal tails of ataxin-2 also have a high content of
proline (179 prolines out of 1090 amino acids, 16.4%).
This property and the low complexity of unstructured
sequence regions may lead to several signicant, but
probably false-positive, hits during a PSI-BLAST search for
homologues of ataxin-2. For instance, despite the use of the
standard low complexity lter, our PSI-BLAST search with
human ataxin-2 homologues found several questionable hits
outside globular domains to homologues of the polyglutamine DRPLA gene product atrophin. For instance,
starting the PSI-BLAST search with an Arabidopsis thaliana
ataxin-2 homologue (SPTrEMBL: Q94AM9), human
atrophin is retrieved in the third iteration with an E-value
of 5 10)11. Conversely, using the rat atrophin homologue
(SPTrEMBL: Q62901) as the start sequence, human ataxin2 was detected in the second iteration with an E-value of
8 10)04.

Fig. 1. Protein architectures of human ataxin-2, its yeast homologue Pbp1, and the P. falciparum homologue PF13_0048 of the decapping enzyme
DCP2 (DCP2_Pf).


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RNA binding of ataxin-2

The Lsm domain of ataxin-2 is typical of RNA-binding Sm
and Sm-like proteins, which often form cyclic 6-, 7- or even
14-oligomers [4143]. Generally, Lsm domain proteins are
involved in a variety of essential RNA processing events
including RNA modication, pre-mRNA splicing, and
mRNA decapping and degradation. Some of them are also
important components of spliceosomal small nuclear ribonucleoproteins (snRNPs).
The LsmAD domain is contained in the Pfam database
with the name Ataxin-2_N and also occurs in another, as
yet uncharacterized Plasmodium falciparum/yoelii yoelii
gene products PF13_0048/PY07327 without an Lsm
domain (Fig. 1). Both Plasmodium gene products have an
additional N-terminal DCP2 domain (also termed box A),
which is always followed by a NUDIX domain [44] in all
known DCP2 homologues. This NUDIX domain constitutes the catalytic subunit of the mRNA decapping
holoenzyme DCP1DCP2 [45,46].
The physiological function of ataxin-2 and closely related
eukaryotic homologues in RNA processing is as yet quite
unexplored [4750]. Interestingly, ataxin-2 has been
observed to interact with A2BP1 (ataxin-2 binding protein
1) [38], whose RNA-binding Caenorhabditis elegans homologue, fox-1, regulates tissue-specic alternative splicing [51].
Disruption of the human A2BP1 gene may cause epilepsy or
mental retardation [52]. In addition, ataxin-2 shows signicant homology to the yeast protein Pbp1 (Pab1/PABPbinding protein 1), which also contains the Lsm and
LsmAD domains; regions outside of these two globular
domains are predicted to be mainly unstructured in Pbp1 as
in ataxin-2.
Although the C-terminal tail of Pbp1 does not contain a
PAM2 motif [39], this yeast protein regulates polyadenylation after pre-mRNA splicing and interacts with the
C-terminal part of the yeast homologue PAB1 of the
human PABP [53]. A2BP1 and PABP are also evolutionarily related and possess RNA recognition motifs [38]. These
observations strongly suggest that ataxin-2 is involved in
similar mRNA processing tasks.
Structural modelling of ataxin-2
First, we compiled a list of ataxin-2 homologues including
the yeast homologue Pbp1 and several Lsm domains of
snRNPs and other Sm and Sm-like proteins from various
species. Then, we assembled a structure-based multiple
sequence alignment of the Lsm domains, crystallographically determined structures of which reveal a close structural
homology between archaeal and eukaryotic proteins
(Fig. 2) [42,43,5465]. This suggests that the function and
the RNA-binding mode of the Lsm domain have been
preserved during evolution.
The RNA-binding Lsm domain is characterized by a
conserved sequence motif consisting of two short segments
known as Sm1 and Sm2, which are separated by a variable
linker [66,67]. The very strong conservation of certain
glycine residues is especially striking and also demonstrates
the evolutionary relationship of ataxin-2 to Lsm domain
proteins. The amide groups of the glycines are known to
stabilize the protein structure when forming hydrogen

Analysis of ataxins 2 and 3 (Eur. J. Biochem. 271) 3157

bonds to adjacent b-strands [55]. The secondary structure

predictions of ataxin-2 and its yeast homologue Pbp1 are
also in good agreement with the known structure of the Lsm
domain as open b-barrel, consisting of an N-terminal
a-helix followed by a strongly bent ve-stranded antiparallel
b-sheet with a 310 helical turn in some cases before the fth
b-strand.
The top two alignment rows in Fig. 2 show human
ataxin-2 aligned with the Pyrococcus abyssi Sm1 protein
(PDB identier 1m8v, chain A), the crystal structure of
which consists of a heptameric ring with a central cavity like
other Lsm domain oligomers [65]. This Sm1 protein
provides the only Lsm domain structure, which is bound
to RNA inside and outside of the doughnut-shaped ring at
an internal and an external binding site. Therefore, we used
this alignment of ataxin-2 to Sm1 to model the 3D structure
of the Lsm domain of ataxin-2 in complex with RNA and
Lsm domains of ataxin-2 protomers (Fig. 3).
Functional analysis of the Lsm domain
We applied the same colour scheme to functionally relevant
residues shown in the multiple sequence alignment and the
3D model of ataxin-2 (Figs 2 and 3). Based on the crystal
structure of Sm1 from P. abyssi bound to uridine heptamers
(U7), we marked several amino acids in Sm1, which are
involved in RNA binding [65] and are mostly physicochemically conserved in ataxin-2 (Sm1/ataxin-2 residue
numbers). The residues forming the internal U7 binding site
are H37/K299, N39/L302 and R63/K330, while ionic
interactions between K22/K284, R63/K330 and D65/S332
stabilize the RNA-binding area. The residues involved in the
external U7 binding site are R4/R266, H10/T272 and Y34/
Y296, stabilized by a hydrogen bond between H10/T272
and Y34/Y296. It is interesting to note that Sm1 from
P. abyssi and from Archaeoglobus fulgidus (PDB identier
1i4k, chain A) share identical RNA-binding residues except
for H10, which is replaced by an asparagine [59,65].
Furthermore, we investigated whether ataxin-2 may also
form oligomers through the Lsm domain. To this end, we
used the detailed crystal structure analyses of the very
similar snRNP heterodimers D1D2 and D3B [55]. Because
of analogous intermolecular interactions in both dimers, we
focused on the complex of D3 with B. This complex is
stabilized mainly by the pairing of the fth b-strand (b5)
from D3 with the fourth b-strand (b4) from B (D3/ataxin-2
B/ataxin-2): R69/V335R73/K330, L71/V337L71/L328,
and L73/F339L69/S326. In addition, two hydrophobic
clusters formed by residues of D3 and B contribute to the
stability of the dimer. The rst cluster includes F70/V336
and I72/Q338 (both in b5 strand) of D3 and F27/Y289
(b2 strand), L67/M324, V70/I327 and L72/L328 (all in b4
strand) of B. The second cluster consists of P6/M267, L10/
L271 (both in a-helix), V18/C279 (b1 strand), L32/F293 (b2
strand), I33/K294 (loop after b2 strand), I68/F334, L71/
V337 and L73/F339 (all in b5 strand) of D3 and I41/L304,
C43/A306 (both in b3), L69/S326 and L71/L328 (both in
b4) of B. Stacking interactions between guanidinium groups
of arginines R69/V335 of D3 and R25/G287 and R49/T312
of B as well as an ionic interaction between E21/Q282 of D3
and R65/S322 of B stabilize the dimer further. However, the
latter salt bridge is not observed in the D1D2 complex

Fig. 2. Structure-based multiple sequence alignment of the Lsm domains of ataxin-2 homologues including the yeast homologue Pbp1 (upper part) with Sm and Sm-like proteins (lower part). The known DSSP
secondary structure assignment of the Sm1 protein from P. abyssi is shown at the top of the alignment (cylinder for a-helix, arrow for b-strand), and the amino acid sequences of crystallographically
determined PDB structures of Lsm domains are underlined accordingly (curled line for a-helix, straight line for b-strand). The corresponding secondary structure predictions for the Lsm domains of ataxin2 and Pbp1 are also given. Physico-chemically similar amino acids are coloured identically. The highly conserved glycines characteristic of Lsm domains are indicated. In the upper part, blue text boxes
point to functionally relevant amino acids forming an internal binding site for uridine heptamers bound to Sm1 from P. abyssi, and green text boxes mark amino acids of the external RNA binding site. In
the lower part, orange text labels annotate how the dimerization of the snRNPs D3 and B is stabilized by intermolecular interactions. PDB identiers and corresponding SPTrEMBL accession numbers for
Lsm proteins are given in Table S2.

3158 M. Albrecht et al. (Eur. J. Biochem. 271)

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Analysis of ataxins 2 and 3 (Eur. J. Biochem. 271) 3159

Fig. 3. 3D model of the Lsm domain of ataxin-2 using three adjacent protomers of the Sm1 protein from P. abyssi as template (PDB identier 1m8v,
chain A, B and G). The model illustrates predicted internal (blue) and external (green) binding sites of ataxin-2 to RNA (grey). a-Helices are in
shown in red, b-strands are shown in cyan. Only functionally relevant residues of the central ataxin-2 protomer are annotated as follows: dark blue
boxes point to residues forming the internal site, and light blue boxes mark amino acids stabilizing the RNA binding area; dark green boxes
highlight residues involved in the external site, and light green ones indicate stabilizing hydrogen bonds.

despite identical amino acids. Altogether, the degree of

conservation of amino acids relevant for heterodimerization
is only moderate, but may still suggest that ataxin-2 may
form Lsm domain oligomers.
Protein architecture of ataxin-3
The longest splice variant of ataxin-3 possesses 376 amino
acids (including 22 glutamines of the polyQ stretch, amino
acid 296317) and an approximate molecular weight of
42 kDa. Ataxin-3 consists of a globular deubiquitinating
N-terminal Josephin domain (amino acid 1170) [68,69] and
a exible C-terminal tail containing two ubiquitin-interacting motifs (UIMs) [70] (also termed LALAL motifs and
PUBs [71], amino acid 223240 and 243260) and the polyQ
region (amino acid 296317) (Fig. 4) [72]. A slightly shorter
alternative splice variant of ataxin-3 with 373 amino acids
has a third UIM (amino acid 334351) at the C-terminus.
An as yet uncharacterized ataxin-3 paralogue on the X
chromosome (sequence identity 70%) is expressed in testis
(ataxin-3t) [10]. The Josephin domain is also found without
a C-terminal tail in other, as yet uncharacterized, proteins
named josephins (Fig. 5) [73].
A highly conserved, putative nuclear localization signal
(NLS) is found upstream of the polyQ stretch (RKRR,
amino acid 282285), which may be bipartite in the
Caenorhabditis elegans homologue of ataxin-3, consisting
of 17 residues (RRDRQKFLERFEKKKEE, amino acid

296312). This NLS follows a potential casein kinase II

(CK-II) phosphorylation site (TSEE, amino acid 277280),
which may determine the rate of the observed ataxin-3
transport into the nucleus [74]. Ataxin-3 may also contain
a nuclear export signal (NES) following the Josephin
domain (ADQLLQMIRV, amino acid 174183) based on
our comparison with a published sequence prole of nuclear
export signals [75]. Furthermore, ataxin-3 contains several
conserved sequence motifs similar to NR- and CoRNRboxes L-x-x-L-L/[IL]-x-x-[IV]-I of transcriptional coactivators and corepressors, respectively [73]. Indeed, ataxin-3
interacts with histones and the histone acetyltransferases
CBP, p300, and PCAF, which work as transcriptional
coactivators. In particular, dependent on these cofactors,
ataxin-3 represses histone acetylation and transcription [76],
and altered protein acetylation has already been implicated
in polyglutamine disease processes [77]. Generally, the
(de-)ubiquitination of histones has been linked to transcriptional regulation [78], which may also explain the observed
interactions of ataxin-3.
Ataxin-3 is evolutionarily conserved in eukaryotes including P. falciparum and plants, but not yeast. The P. falciparum homologue PFL1295w of ataxin-3 (ataxin-3_Pf),
whose gene expression is upregulated similarly to the
P. falciparum josephin homologue PF11_0125 in gametocytes [7981], constitutes an exception because it has only
the second UIM conserved (amino acid 250267) and has
an additional ubiquitin-like UBX domain [8285] at the


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3160 M. Albrecht et al. (Eur. J. Biochem. 271)

Fig. 4. Protein architectures of human ataxin-3, its P. falciparum

homologue PFL1295w (ataxin-3_Pf), and human josephin 1.

C-terminus (amino acid 271381) instead of the polyQcontaining region [69]. Like human ataxin-3, this ataxin-3
homologue PFL1295w also has a potential casein kinase II
phosphorylation site (TSDE, amino acid 278281) close to
basic amino acids, which can be indicative of an NLS
(KKIH, amino acid 293296) near the N-terminus of the
UBX domain. In contrast, the prediction server PSORT II
returns another region inside the UBX domain as a possible
NLS (PRRK, amino acid 339342). It is unclear which NLS
motif may be functionally more relevant because both
NLS motifs correspond to amino acids at solvent exposed
N-termini of the second and fourth b-strand in the crystal
structure of the UBX domain of the cofactor p47 (PDB
identier 1s3s) [86]. Similar to the P. falciparum homologue,
the Cryptosporidium parvum homologue of ataxin-3 also
possesses only one UIM motif (amino acid 266283) and a
C-terminal UBX domain (amino acid 288397) instead of a
polyQ region.
Ubiquitin binding of ataxin-3
Ubiquitination fullls many cellular functions in cytoplasmic trafcking, guiding specic proteins through the
endocytic pathways, and targeting proteins to the protea-

some [84,8793]. Above all, the ubiquitinproteasomal

pathway is involved in processing mutant or damaged
proteins that cause neurodegenerative diseases. The small
ubiquitin protein can be covalently linked to other proteins
as single molecule or polyubiquitin chain.
Recently, the two UIMs between the Josephin domain
and the polyQ stretch of ataxin-3 have been shown to be
capable of binding tetraubiquitin and polyubiquitinated
proteins [68,9497]. In our previous study, we used the
C-terminal ANTH domain extension, which consists of an
antiparallel three-helix bundle, to model the structure of the
UIMs in the C-terminal tail of ataxin-3 [73]. In fact, novel
structure determinations have shown that UIM peptides are
a-helices and can form helix bundles in the crystal structure
[98]. In contrast, the NMR solution structures of UIM
peptides reveal that they are single amphiphatic a-helices
connected by unstructured linkers [99,100]. The latter
observation is in agreement with the observed exibility of
the C-terminal tail of ataxin-3 [72].
Furthermore, the ANTH domain itself is evolutionarily,
structurally, and functionally related to a VHS domain
[101]. Lately, the structure of the GAT (GGAs and Tom1)
domain directly following the VHS domain of Tom1 and
GGAs (Golgi-associate, c-adaptin ear-containing, Arfbinding proteins) was determined crystallographically
[102105]. The GAT domain contains a three-helix bundle,
which we found to superimpose very well with the helical
bundle of the C-terminal ANTH domain extension (RMSD
3.1 A, PDB identiers 1o3x and 1hx8, A chains).
Interestingly, the GAT domains of GGAs and Tom1
have been reported to interact with ubiquitin [106108].
The corresponding ubiquitin binding site was located to the
third a-helix of the GAT three-helix bundle, and hydrophobic amino acids like leucines are important for the
interaction (Fig. 5). The same residue type also plays an
essential role in binding ubiquitin to the UIM a-helix [98
100] and the third a-helix of the helical bundle in the
homologous CUE and UBA domains [109]. However, the
sequence similarity is quite low, and thus it is difcult to
deduce an evolutionary relationship, although the ubiquitin
binding sequence of GGAs and Tom1 resembles a
noncanonical UIM whose, otherwise strictly conserved,
serine residue is replaced by an asparagine except in case of
human GGA3 (Fig. 5).
Further interaction partners of ataxin-3
It has been shown that ataxin-3 interacts with the ubiquitinlike (UBL) domain of the homologous ubiquitin- and
proteasome-binding factors hHR23A and hHR23B, whose
yeast orthologue is Rad23 [96,110112]. The latter factors
are also involved in the nucleotide excision repair pathway
by targeting the ubiquitinated nucleotide excision repair
factor XPC/Rad4 to the proteasome [113]. Their UBL
domain binds to a UIM helix of the 26S proteasome subunit
S5a, and this interaction disrupts the interdomain contacts
between the N-terminal ubiquitin-mimicking UBL domain
and the two C-terminal ubiquitin-binding UBA domains,
thereby inducing the change from a closed to an open
protein conformation [109,111,114,115]. Rad23 and the
yeast orthologue Rpn10 of S5a serve as alternative ubiquitin
receptors for the proteasome [116], and the UBA domains


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Analysis of ataxins 2 and 3 (Eur. J. Biochem. 271) 3161

Fig. 5. Multiple sequence alignment of UIM peptides, divided into

groups by horizontal lines from top to bottom: UIM sequences of the
Pfam seed alignment including rst, second, and third UIMs of ataxin-3
homologues, UIM-like peptides from GGAs and Tom1, and related
AP180 sequences. The latter are derived from the 3D structure superposition of the GAT domain of human GGA1 with the AP180 extensions from Rattus norvegicus and D. melanogaster (PDB identiers 1hf8
and 1hx8, respectively). The second group of UIMs in ataxin-3 homologues also includes the similar N-terminal a-helix of the UBX domain
extension of p47 (PDB identier 1s3s). For each group, amino acids in
alignment columns with a majority of identical residues are printed on a
black background, and similar amino acids are highlighted in grey.

of Rad23 inhibit proteasome-catalysed proteolysis by

sequestering Lys48-linked polyubiquitin chains [117,118].
In particular, the NMR solution structures of the UBL
domain of hHR23A/B bound to a UIM peptide of S5a
[99,119] could be used to model the complex of hHR23A/B
and ataxin-3. Similarly, the complex of a UIM of ataxin-3
with ubiquitin could be modelled based on the NMR
solution structure of the UIM of the Vps27 protein bound
to ubiquitin [100].
The C-terminal region of ataxin-3 including the polyQ
region interacts with the N-terminal cofactor/substratebinding adaptor domain of the valosin-containing protein
VCP/p97/Cdc48/VAT/ter94 [96,120123]. VCP is an
important multifunctional AAA+ ATPase with two Cterminal ATPase domains after the adaptor domain, which
provide the energy for major conformational changes [124].
VCP forms hexamers and works as molecular chaperone
involved in a variety of intracellular functions including cell
cycle progression, membrane fusion, vesicle-mediated transport, transcription activation, apoptosis prevention, and
ubiquitin-proteasome degradation, modulating polyglutamine-induced neurodegeneration [96,120123,125127].
VCP binds the ubiquitin E3 ligase and the chain assembly
factor UFD2a/E4B, which is a U box homologue of yeast
Ufd2 [128], and interacts with and regulates the degradation
of the proteasome-associated ataxin-3, forming a trimeric
complex of ataxin-3, VCP, and UFD2a [96,127,129131].
Interestingly, Ufd2 binds the UBL domain of Rad23 and
competes with Rad23 for binding to the Rpn1 proteasome
subunit, while the N-terminal UBL domain of the ubiquitin
C-terminal hydrolase Ubp6 interacts with Rpn1 without
competition with Rad23 [116,132].
Furthermore, VCP also binds the C-terminal UBX
domain of the membrane fusion adaptor p47/SHP1/EYC/
Ubx3 [85,86,133], which consists of three domains UBASEP-UBX [134]. The crystallographically determined complex of the N-terminal adaptor domain of VCP with this
UBX domain (PDB identier 1s3s) indicates the interacting
residues [86] and could be used to model the putative
complex of VCP with the C-terminal UBX domain of the
ataxin-3 homologue from P. falciparum (ataxin-3_Pf). Like
the UBX domain of p47, ataxin-3_Pf contains the conserved
loop that is essential for an interaction with VCP because it
inserts into a hydrophobic pocket of VCP [86]. The UBX
domain structure of p47 is extended at its N-terminus by a
disordered peptide structure and an additional a-helix of as
yet unknown functional relevance [86]. The length of this
a-helix is similar to a UIM a-helix (Fig. 5), and such a UIM

also precedes the UBX domain of ataxin-3_Pf. Therefore,

this a-helix of p47 might be related to the second UIM in
ataxin-3 homologues (recall that the rst UIM is missing in
ataxin-3_Pf). In addition, the arrangement of one UIM
helix followed by a C-terminal UBX domain is also found in
the cofactor Ubx2 with domain architecture UBA-UAS-

3162 M. Albrecht et al. (Eur. J. Biochem. 271)

UIM-UBX [133]. The UIM of Ubx2 binds ubiquitin chains,

and the UBX domain interacts with VCP. Thus the same
interactions can be expected for ataxin-3_Pf.
The C-terminal, presumably VCP-binding, UBX domain
of ataxin-3_Pf appears to correspond to the VCP-binding
C-terminal part of human ataxin-3, which follows the
second UIM and includes the polyQ region [120,123,131].
In addition, the polyQ tract of ataxin-3 has been shown to
be indispensable for the interaction with VCP, and its length
correlates with the strength of the interaction. These observations raise the question how human ataxin-3 binds VCP
in contrast to its P. falciparum homologue. This is particularly interesting because VCP may suppress polyQ induced
neurodegeneration, and mutations in VCP have been
observed to cause cytoplasmic vacuoles followed by cell
death because of a dysfunctional second ATPase domain
and inclusion body formation [120123,127,135,136]. We
also observed that all VCP sequence variations associated
with Paget disease of bone and frontotemporal dementia
(IBMPFD) [135] are not located in the binding interface of a
UBX domain with the N-terminal adaptor domain of VCP,
but are involved in interactions between protein regions
(for details see the online supplement). Therefore, motions
of the adaptor domain, which are essential for proper VCP
function [124,127], may be impaired by IBMPFD-associated mutations.
According to a recent yeast-2-hybrid screen [137], a
josephin homologue from Drosophila melanogaster
(CG3781) on the X chromosome interacts with the heat
shock protein HSP60b (CG2830), which is involved in
spermatogenesis [138,139], suppresses ubiquitination [140]
and associates with 38 further proteins including a ubiquitin
E3 ligase, but no other deubiquitinating enzyme except
josephin (CG8184). Interestingly, HSP40 and HSP70 chaperones have already been observed to associate with VCP,
and they also colocalize with intranuclear ataxin-3 aggregates and may play an important role in the disease process
and the impairment of the ubiquitin-proteasome system
[121,141149].
Structural modelling of the Josephin domain
Recently, it has been observed that the Josephin domain
contains highly conserved amino acids reminiscent of the
catalytic residues of a deubiquitinating cysteine protease
[69], and rst experimental results support this function
hypothesis [68]: decrease of polyubiquitination of 125Ilabelled lysozyme by removal of ubiquitin, cleavage of the
ubiquitin protease substrate ubiquitin-AMC, and binding of
the specic ubiquitin protease inhibitor ubiquitin-aldehyde
(Ubal). Mutating the catalytic cysteine in ataxin-3 inhibits
these functions [68].
Previously, we modeled the 3D structure of ataxin-3
based on the ANTH domain [150] of the adaptin AP180
as structural template [73]. However, this prediction has to
be revised with regard to the N-terminal Josephin domain
because of the identied cysteine protease signature [69].
In contrast to our previous prediction [73], which relied
on the secondary structure prediction from a single server,
we now formed the consensus result of the three state-ofthe-art secondary structure prediction servers PSIPRED,
SAM-T99, and SSpro2. All three online servers basically

FEBS 2004

returned the same secondary structure for human ataxin-3

and josephin 1, resulting in a much more reliable
secondary structure prediction of b-strands besides a-helices. We propose that the increased accuracy of this
prediction is due, at least in part, to a substantial growth
of protein sequence and structure databases. The predicted b-strands in the Josephin domain corroborate a
cysteine protease fold of deubiquitinating enzymes
(DUBs) and do not support the ANTH domain structure
consisting solely of a-helices. In hindsight, the fold
recognition methods applied in the past to predict the
structure of ataxin-3 may have been misguided by the
pronounced prediction of a-helices only.
DUBs process ubiquitin proteolytically at the C-terminus
and can be divided into at least two evolutionarily related
families of cysteine proteases, UBPs (ubiquitin-specic
proteases) and UCHs (ubiquitin C-terminal hydrolases)
[151,152]. However, new ubiquitin-specic families such as
otubains (OTU) and JAMMs with low sequence similarity
to known DUBs are still being discovered [151]. A
consensus of fold recognition servers now selects both
available UCH domain structures of human UCH-L3 [153]
and yeast YUH1 [154], which superimpose with a low
RMSD of 2.0 A (PDB identiers 1uch and 1cmxA,
respectively), as best modelling templates with a moderate
condence score for human josephin 1, but still with only a
weak score for ataxin-3. The pairwise sequencestructure
alignments returned by the structure prediction servers for
3D modelling differ mainly in the central part of the
Josephin domain (amino acid 47117 in ataxin-3) aligned to
DUBs. This nding underpins the distant relationship of the
Josephin domain to known DUBs. The central part does
not contain catalytic residues and is thus less conserved,
containing insertions of variable length and structure in
other cysteine proteases [155].
Based on a multiple sequence alignment of Josephin
domain homologues (Fig. 6), we used the crystallographically determined structure of YUH1 bound to the ubiquitinlike inhibitor Ubal (PDB identier 1cmx, chains A and B,
respectively) to model the tertiary structure of the Josephin
domain of ataxin-3 in complex with Ubal (Fig. 7). Thus, the
structure of ataxin-3 is predicted to be distinct from the
ngerpalmthumb architecture of UBPs such as USP7/
HAUSP [156]. Because of the low degree of conservation in
the central part, we believe that ataxin-3 and josephin 1
adopt slightly different structures in this part, which are not
very similar to YUH1. In addition, we observed that the
Josephin domain also resembles the OTU domain because
both have a highly conserved histidine three residues
downstream of the catalytic cysteine. Interestingly, like
ataxin-3, the deubiquitinating OTU domain protein
VCIP135 interacts with the N-terminal adaptor domain of
VCP through the C-terminal tail including a UBL domain
and dissociates p47 from the complex with VCP during
ATP hydrolysis of VCP [157,158]. This observation also
indicates a close functional relationship of the homologous
ubiquitin-like UBL and UBX domains.
Functional analysis of the Josephin domain
The active site of UCHs is divided into two parts as
follows (YUH1/ataxin-3 residue numbers) [153,154]: The


FEBS 2004

Analysis of ataxins 2 and 3 (Eur. J. Biochem. 271) 3163

Fig. 6. Structure-based multiple sequence alignment of the Josephin domains of ataxin-3 homologues with the crystallographically determined UCH
domains of human UCH-L3 and yeast YUH1. The known DSSP secondary structure assignments of UCH-L3 and YUH1 are shown at the top of
the alignment (curled lines for a-helix, arrows for b-strands). The corresponding consensus secondary structure predictions for human ataxin-3 and
josephin 1 are also depicted. Alignment columns with identical residues are highlighted in purple-coloured boxes, those with more than 50%
physico-chemically similar amino acids in yellow boxes (bold-printed letters). Text labels (including UCH-L3/YUH1 and ataxin-3/josephin 1
residue numbers) point to catalytic residues (four grey-shaded boxes) and to other highly conserved amino acids in the Josephin domain. The PDB/
SPTrEMBL identiers of UCH-L3 and YUH1 are 1uch/P15374 and 1cmxA/P35127, respectively. NCBI or Ensembl accession numbers for
Josephin domain homologues are given in Table S3.

N-terminal part consists of a glutamine (Q84/Q9) upstream

of a cysteine (C90/C14), both of which form an oxyanion
hole to accommodate the negative charge on the substrate
carbonyl oxygen during catalysis. The C-terminal part
contains a histidine (H166/H119), which is thought to be
deprotonated, and an asparagine or aspartate (D181/N134),
both of which activate the side chain of the cysteine to

unleash a nucleophilic attack on the carbonyl carbon atom

of the scissile peptide bond. The cysteine, histidine, and
asparagine/aspartate constitute the catalytic triad characteristic of cysteine proteases such as papain.
While all four discussed catalytic residues are strictly
conserved in the Josephin domain (Fig. 6), a functionally relevant disordered loop (E144N164/V79Q100)

3164 M. Albrecht et al. (Eur. J. Biochem. 271)

FEBS 2004

Fig. 6. (Continued).

positioned over the catalytic cleft is aligned in the less

conserved central part. This loop maintains an inaccessible
active site, but becomes ordered upon binding of Ubal [154].
Therefore, it may control substrate specicity together with
further strongly conserved amino acids such as N88/L13,
which forms hydrogen bonds with main chain groups of the
loop, and Y167/W120 next to the catalytic histidine [154].
Unfortunately, the structure of the central part and the
loop function remains unclear for the Josephin domain
because of insufcient sequence similarity to UCHs. The
Josephin domain is also missing the N-terminal extensions
of UCHs, which are involved in substrate recognition [154].
In addition, a functional relevance of a second strictly

conserved histidine H17, two highly conserved asparagines

N20 and N21, and another identical glutamine Q24
downstream of the catalytic cysteine C14 cannot be derived
either from the structural model of the Josephin domain
(Figs 6 and 7). However, considering their distance from the
active site and location inside the protein, they may be solely
important for the stability of the domain fold. This may also
hold true for the strictly conserved S135 and P140 after the
catalytically active N134. In contrast, it is easy to interpret
an alternative splice variant of ataxin-3 [10], which consists
of a deletion of the residues from E10 to Q64 including
the catalytic cysteine and thus cannot possess proteolytic
activity.


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Analysis of ataxins 2 and 3 (Eur. J. Biochem. 271) 3165

Fig. 7. 3D model of the deubiquitinating Josephin domain of ataxin-3 using the structure of yeast YUH1 bound to the ubiquitin-like inhibitor Ubal (in
CPK view mode) as template (PDB identier 1cmx, chains A and B, respectively). Grey-shaded text labels indicate the four catalytic residues (balland-stick view) forming the active site of the ubiquitin hydrolase. The remaining text boxes point to other residues, which are highly conserved in the
Josephin domain. Residues are coloured in agreement with the alignment columns in Fig. 6. The N-terminal extension of YUH1, which is missing
in ataxin-3 homologues, is depicted in the background as thin dark brown protein backbone only. The less conserved central part of ataxin-3 is
shown in green; it could not be modelled reliably using YUH1 as template because of low sequence similarity.

Comparison to other polyQ proteins

Conclusions

The polyQ stretch of both ataxin-2 and ataxin-3 lies in

sequence regions whose degree of conservation is very low
in contrast to the globular domains and which are
predicted to be intrinsically unstructured. This prediction
has been conrmed experimentally for ataxin-3 [72], and
polyQ tracts themselves also adopt a random coil
conformation [159]. So we decided to investigate other
polyglutamine disease proteins such as ataxin-1 (SCA1),
ataxin-7 (SCA7), atrophin (DRPLA) and huntingtin (HD)
as to whether their polyQ regions are also predicted to be
surrounded by disordered structure. For this purpose, we
used several online prediction servers (DisEMBL, DISOPRED, GlobPlot, NORSp, PONDR), which consensus
basically indicates that the polyQ tracts are generally
located in unstructured regions (Table S4). This is also in
agreement with secondary structure prediction results,
which do not indicate globular domains consisting of
a-helices or b-strands (data not shown), and other
computational predictions of locally unfolded regions
[160]. Therefore, it is not surprising that mutant polyglutamine proteins can readily form aggregates via the
solvent-exposed polyQ region.

We presented a detailed analysis of ataxin-2 homologues

including the yeast homologue Pbp1, using a structurebased multiple sequence alignment of Sm and Sm-like
proteins and a 3D model of the Lsm domain of ataxin-2.
Our comparison revealed a high degree of conservation of
chemical properties for RNA-binding residues in the aligned
Lsm domains in general and between Sm1 from P. abyssi
and human ataxin-2 in particular. Based on this observation, we propose that ataxin-2 is capable of binding RNA
by the identied residues. Therefore, an essential function of
ataxin-2 homologues in RNA processing should be
explored experimentally and could implicate the regulation
of polyadenylation of mRNA as it is known for Pbp1. In
addition, the similarity of amino acids involved in the
formation of Lsm domain oligomers as derived from the
D1D2 and D3B heterodimers may suggest that ataxin-2
may also form such complexes.
Our structural model of the Josephin domain of ataxin-3
conrms the evolutionary relationship with deubiquitinating cysteine proteases of the UCH family. Interestingly, this
relates ataxin-3 to another ubiquitin hydrolase termed
USP14, which is involved in synaptic dysfunction in ataxic


FEBS 2004

3166 M. Albrecht et al. (Eur. J. Biochem. 271)

mice [161,162]. Moreover, the polyglutamine disease protein

ataxin-1 interacts with the ubiquitin-specic protease USP7/
HAUSP, and the length of the polyQ region inuences the
strength of the interaction [163]. Unfortunately, the central
part of the Josephin domain is difcult to model because of
low sequence similarity. Therefore, it cannot be deduced
whether the ataxin-3 mechanism of ubiquitin recognition
works similarly to UCHs.
It is striking that both human ataxin-3 and its P. falciparum homologue ataxin-3_Pf can bind the N-terminal
adaptor domain of the molecular chaperone VCP at their
C-termini, although they differ considerably and an evolutionary relationship is not apparent: ataxin-3 contains the
polyQ region, but the P. falciparum homologue has a
ubiquitin-like UBX domain. Another open question is
whether a deubiquitinating analogue of ataxin-3 exists in
yeast, since the Josephin domain is not found in any yeast
protein, but complexes of the ataxin-3 and proteasome
binding proteins hHR23A/B, VCP and UFD2a are conserved in yeast. Generally, it remains to be seen how the
normal functions of ataxin-2 and ataxin-3 are affected in
mutant proteins with an expanded polyglutamine tract.

11.

12.

13.

14.

15.

16.

Acknowledgements
Part of this research was funded by the German Research Foundation
(DFG) under contract no. LE 491/141, by the Federal Ministry of
Education and Research (BMBF) under contract no. 01gs0115-NVS02T12, and by the European Commission through the EUROSCA
project under contract no. LSHM-CT-2004-503304.

17.

18.

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Supplementary material
The following material is available from
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ejb/ejb4245/ejb4245sm.htm
Appendix. Supplementary online material.