Tài liệu Báo cáo khoa học: Structural bases for recognition of Anp32⁄LANP proteins doc

Structural bases for recognition of Anp32⁄ LANP proteins
Cesira de Chiara, Rajesh P. Menon and Annalisa Pastore
National Institute for Medical Research, The Ridgeway, London, UK
The leucine-rich repeat acidic nuclear protein (Anp32a ⁄
LANP) is a member of the Anp32 family of acidic
nuclear evolutionarily-conserved phosphoproteins,
which present a broad range of activities [1]. They are
characterized by the presence of a highly conserved
N-terminal domain containing leucine-rich repeats
(LRRs), motifs known to mediate protein–protein inter-
actions, and of a C-terminal low-complexity region,
mainly composed of polyglutamates.
Since their first description in the neoplastic B-lym-
phoblastoid cell line and their reported association
with proliferation [2], several Anp32a homologs, all
derived from a common ancestor gene by subsequent
duplication events, have been isolated in different tis-
sues and differently named [1]. Members of the Anp32
family are widely recognized as nucleo-cytoplasmic
shuttling phosphoproteins that are implicated in differ-
ent signaling pathways and in a number of important
cellular processes, which include cell proliferation, dif-
ferentiation, caspase-dependent and caspase-indepen-
dent apoptosis, tumor suppression, regulation of
mRNA trafficking and stability, histone acetyltransfer-
ase inhibition, and regulation of microtubule-based
functions [1,3].
The diverse activities of Anp32 proteins are achieved
through an articulated network of interactions with
several cellular partners. Among them, two proteins
are of particular relevance from the clinical point of

view. Anp32 proteins are powerful inhibitors of phos-
phatase 2A (PP2A), a major serine ⁄ threonine phospha-
tase involved in many essential aspects of cellular
function [4–8]. PP2A, which is considered to be the
principal guardian against cancerogenic transforma-
tion, is a dynamic, structurally diverse molecule found
in several different complexes and able to react to a
plethora of signals [9–12]. The N-terminal LRR
Keywords
ataxin 1; leucine-rich repeats; NMR; PP2A
inhibitor; structure
Correspondence
A. Pastore, National Institute for Medical
Research, The Ridgeway, London NW7
1AA, UK
Fax: +44 208 906 4477
Tel: +44 208 959 3666
E-mail:
(Received 13 January 2008, revised 3 March
2008, accepted 14 March 2008)
doi:10.1111/j.1742-4658.2008.06403.x
The leucine-rich repeat acidic nuclear protein (Anp32a ⁄ LANP) belongs to
a family of evolutionarily-conserved phosphoproteins involved in a com-
plex network of protein–protein interactions. In an effort to understand the
cellular role, we have investigated the mode of interaction of Anp32a with
its partners. As a prerequisite, we solved the structure in solution of the
evolutionarily conserved N-terminal leucine-rich repeat (LRR) domain and
modeled its interactions with other proteins, taking PP2A as a paradig-
matic example. The interaction between the Anp32a LRR domain and the
AXH domain of ataxin-1 was probed experimentally. The two isolated and

unmodified domains bind with very weak (millimolar) affinity, thus sug-
gesting the necessity either for an additional partner (e.g. other regions of
either or both proteins or a third molecule) or for a post-translational
modification. Finally, we identified by two-hybrid screening a new partner
of the LRR domain, i.e. the microtubule plus-end tracking protein
Clip 170 ⁄ Restin, known to regulate the dynamic properties of microtubules
and to be associated with severe human pathologies.
Abbreviations
Anp32a ⁄ LANP, leucine-rich repeat acidic nuclear protein; Atx1, ataxin-1; AXH, ataxin-1 homology; Gal-X, 5-bromo-4-chloroindol-3-yl
b-
D-galactoside; GST, glutathione S-transferase; HSQC, heteronuclear single quantum coherence; LRR, leucine-rich repeat; MAP,
microtubule-associated protein; PP2A, phosphatase 2A; RDC, residual dipolar coupling; SCA1, spinocerebellar ataxia 1; TCEP,
Tris(2-carboxyethyl)phosphine; +TIP, plus-end tracking proteins.
2548 FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS
domain of Anp32 binds and strongly inhibits the
enzyme catalytic subunit PP2A-C [3–7], whose struc-
ture in a heterotrimeric complex with the scaffolding
A subunit and the regulatory B¢⁄B56 ⁄ PR61 subunit
was solved recently [13,14]. Although the role of phos-
phorylation in recognition remains debatable, interac-
tion between Anp32a and PP2A-C has been
independently confirmed by high-throughput yeast
two-hybrid screening [15].
Involvement of Anp32 in spinocerebellar ataxia
type 1 (SCA1) pathogenesis was also suggested, on the
basis of the observation of an interaction with the
SCA1 gene product ataxin-1 (Atx1) [16]. This protein
belongs to a family involved in neurodegenerative dis-
eases caused by anomalous expansion of polyQ tracts
[17]. In SCA1, expanded Atx1 forms nuclear inclusions

that are associated with cell death. Immunofluores-
cence studies demonstrated that Anp32a and Atx1
colocalize in nuclear matrix-associated subnuclear
structures. The interaction was mapped onto the LRR
and AXH domain of Anp32 and Atx1 respectively,
and was shown to be stronger for expanded Atx1
[16,18]. The temporal and cell-specific expression
pattern of Anp32 in cerebellar Purkinje cells, the
primary site of pathology in SCA1, as well as its
enhanced interaction with mutant Atx1, have sug-
gested a role for Anp32a in SCA1 pathogenesis.
Despite the importance of molecular interactions
for the presumed cellular functions of Anp32a, very
little is known about their structural bases. The struc-
ture of the Anp32 LRR domain was first predicted
by homology [1] and more recently solved by X-ray
crystallography [19]. Interestingly, although both
reports described the domain as being formed by tan-
dem LRRs, the structures differed in the number of
repeats. This information is not just academic, as
these details would allow accurate definition of the
domain boundaries and help our understanding of
how interactions could take place in different regions
of the molecule.
As part of a long-term function-oriented structural
effort aimed at understanding the molecular bases of
polyQ disease proteins, we report here a study of the
structural determinants for the interactions of Anp32a
with other partners. As a prerequisite for binding stud-
ies, we first solved the structure in solution of Anp32

by NMR spectroscopy. This technique, which does not
need crystallization, also provides a powerful and flexi-
ble method for mapping binding interfaces. Our struc-
ture, as described in the following sections, reveals the
presence of two extra N-terminal LRR motifs not
observed in the crystal, and allows accurate definition
of the C-terminal domain boundary. Experimental
determination of the dynamic features of the domain
in solution, together with a comparison with the struc-
ture of the spliceosomal U2A¢ in complex with U2B¢¢
[20], suggests new insights into the mechanism of
Anp32 LRR–protein recognition. By a combination of
chemical shift perturbation techniques, molecular
docking and two-hybrid screening, we also probed the
interaction with Atx1 and PP2A, and identified a new
partner of the Anp32a LRR domain.
Results
Description of the Anp32a LRR domain structure
in solution
The construct used for structure determination covers
residues 1–164 of the mouse Anp32a sequence [21].
These boundaries were chosen to include the sequence
up to the beginning of the acidic repeats, where
sequence conservation breaks down (data not shown).
The resulting sample was stable and well behaved, pro-
viding NMR spectra typical of a folded monodisperse
globular domain. The final representative family of the
10 lowest-energy structures after water refinement
could be superimposed on the average structure
with overall rmsd values of 0.71 ± 0.15 A

˚
and
1.16 ± 0.16 A
˚
, for backbone and heavy atoms respec-
tively, in region 3–154 (Fig. 1). The structure was
solved at high precision and has an excellent whatif
score (Table 1).
The domain topology (h
1
h
2
b
1
b
2
b
3
h
3
b
4
h
4
b
5
h
5
h
6

b
6
b
7
b
8
) shows the secondary structure elements spatially
arranged in the typical right-handed solenoid, which
forms a curved horse-shoe fold. A canonical parallel
b-sheet is present on the concave side, whereas the
convex surface contains both well-defined but irregular
secondary structure elements (in the first and second
repeats) and helical regions. Among these, h
1
and h
6
are regular a-helices whereas h
2
,h
3
,h
4
and h
5
share
features of 3
10
-helices. A search for tertiary structure
similarity performed by dali [22] indicates that the
Anp32a LRR domain structure belongs to the SDS22-

like LRR subfamily [23].
Comparison with other Anp32 structures
The Anp32a LRR domain is composed of five com-
plete LRRs flanked by an a-helix at the N-terminus
and by the C-terminal flanking motif termed LRRcap
(SMART accession number SM00446), so far identi-
fied in several ‘SDS22-like and typical’ LRR-contain-
ing proteins, such as U2A¢, TAP, RabGGT, and
dynein LC1 [23]. The Anp32a LRRcap motif spans
C. de Chiara et al. Study of the interactions of the Anp32a LRR module
FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS 2549
residues Leu128 to Asp146, and includes h
6
, which
belongs to the fifth LRR, and the short strand b
6
,
which runs parallel to b
5
and is antiparallel to b
7
. The
solution and the crystal structures of the Anp32a LRR
domain superimpose with a 1.1 A
˚
rmsd over the back-
bone atoms of the overlapping region 1–149
(Fig. 2A,B). Despite the structural similarity, only four
repeats (44–65, 66–89, 90–114 and 115–138 in our
structure) were identified in the crystal structure [19],

whereas the first repeat (residues 19–43 in our struc-
ture) was considered by these authors to be an
N-CAP. The presence of the first N-terminal LRR had
also not been predicted [1], probably because of the
low sequence conservation in this region. In our opin-
ion, this region constitutes instead a bona fide full
repeat.
Residues 147–149, which are truncated in the crystal
structure, form a b-hairpin (b
7
) with the strand 143–
145 (b
6
). This region shares a remarkable similarity
with U2A¢, the two protein with the highest structural
homology: the two proteins can be superimposed with
2A
˚
rmsd as calculated over 140 residues and have a
dali z score of 17 (Fig. 2A,C). Although mainly
unstructured, a short additional strand C-terminal to
the hairpin (b
8
) is present in some of the NMR struc-
tures in region 149–164 (residues 151–153). This region,
which constitutes the linker between the LRR domain
and the acidic repeats, is thought to be involved in
interactions with the INHAT complex and the phos-
phorylation-dependent tumor suppressive ⁄ proapoptotic
activity, which have been mapped to residues 150–180

and 150–174, respectively [15,24,25]. Interestingly, this
tail contains one of the two CK2 phosphorylation
motifs (158–161) that have been proved to be natively
phosphorylated [26], out of the four putative sites
predicted by prosite [27]. As expected, the structure of
this region is flexible and is likely to be involved in the
regulation of phosphorylation-dependent functions of
Anp32a [1,3].
Probing the dynamics in solution of the Anp32a
LRR domain
1
H–
15
N relaxation studies were carried out to assess the
dynamic properties of the Anp32a LRR domain
(Fig. 3). The correlation time, as estimated from the T
1
and T
2
relaxation data using the model-free approach
[28], is 12.5 ± 0.1 ns at 27 °C, a value within the range
expected for a single monomeric species of this size in
solution [29]. The flat profile of the relaxation parame-
ters along the sequence indicates that, with the excep-
tion of the first two N-terminal amino acids and of the
C-terminal tail (Ala155–Val164), the structure is rigid
and compact, which is in good agreement with what is
observed from the local rmsd values and the residual
dipolar coupling (RDC) values. Of the seven resi-
dues whose amide connectivities are missing in the

1
H–
15
N heteronuclear single quantum coherence
(HSQC) spectrum at pH 7 (Met3, Asp4, Ile30, Glu31,
Ile34, Glu35 and Val52) the last five belong to regions
without a regular secondary structure. All seven resi-
dues, including Met3 and Asp4 at the N-terminus of
h
1
, cluster together in the structure, suggesting that
they experience chemical or conformational exchange.
The C-terminus is unstructured and highly mobile
approximately from residue 154 onwards.
It is also interesting to note a clear correlation
between T
1
and RDC values and the secondary struc-
ture: the concave b-sheet is characterized by shorter
T
1
and positive RDC values, the latter indicating that
A
B
Fig. 1. Solution structure of the LRR
domain of murine Anp32a. (A) NMR bundle
of the 10 best structures in terms of
energy. (B) Average structure as obtained
by the
WHEATSHEAF algorithm [62]. Two

orthogonal views are shown.
Study of the interactions of the Anp32a LRR module C. de Chiara et al.
2550 FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS
the corresponding residues are oriented parallel to the
external magnetic field B
0
when in the anisotropic
medium [30]. Conversely, the NH vectors in the long
helices (h
1
,h
3
,h
4
, and h
6
) running approximately
parallel to each other are mainly perpendicular to the
b-sheet vectors and, therefore, to B
0
in the aligned
medium.
These results suggest that interactions with other
molecules involving the LRR domain are not medi-
ated by an induced fit mechanism but by semirigid
docking of the partners onto the surface of the
Anp32a LRR domain. Interactions with the INHAT
complex, mapped to the flexible C-terminus, may
induce structuring and stiffening of this region.
Modeling the interaction of Anp32a with other

proteins on the basis of the U2A¢–U2B¢¢ structure
The structural similarity with U2A¢, whose structure is
known in a complex with its target U2B¢¢ [20], may
provide valuable hints on how Anp32 interacts with its
partners. We therefore analyzed this complex and
compared its features with those of our structure. Rec-
ognition of the two molecules occurs by fitting a helix
of U2B¢¢ (residues 25–35) into the concave surface of
the U2A¢ LRR (Fig. 4). The size complementarity is
almost perfect. The nearby N-terminal b-hairpin of
Table 1. Structural statistics for the calculations of the Anp32a
LRR domain.
Final NMR restraints
Total distance restraints
a
5151
Unambiguous ⁄ ambiguous 3774 ⁄ 1376
Intraresidue 2021
Sequential 1075
Medium (residue i to i + j, j = 1–4) 663
Long-range (residue i to i + j, j > 4) 1392
Dihedral angle restraints
b
F 89
w 89
1
D
NH
RDC 92
Hydrogen bonds 20

Deviation from idealized geometry
Bond lengths (A
˚
) 0.003 ± 0.000
Bond angles (°) 0.503 ± 0.011
Improper dihedrals (°) 1.442 ± 0.073
Restraint violations
Distance restraint violation > 0.5 A
˚
0
Dihedral restraint violation > 5° 0
Coordinate precision (A
˚
) with respect to the mean structure
Backbone of structured regions
c
0.71 ± 0.15
Heavy atoms of structured regions
c
1.16 ± 0.16
WHATIF quality check
d
First-generation packing quality 0.01
Second-generation packing quality )2.44
Ramachandran plot appearance )2.77
v1–v2 rotamer normality )0.12
Backbone conformation )1.69
Procheck Ramachandran statistics (%)
Most favored region 75.1
Additional allowed regions 23.8

Generously allowed regions 0.4
Disallowed regions 0.8
a
Calculated for the 10 lowest-energy structures after water refine-
ment.
b
Derived from
3
J(HN, Ha) coupling constants and TALOS [48].
c
Calculated for residues 3–154. The more positive the score, the
better it is. Problematic structures typically have scores around )3.
Wrong structures have scores lower than )3.
d
Calculated for resi-
dues 1–154.
A
B
C
Fig. 2. Comparison between the NMR (A) and the X-ray (B) struc-
tures of the Anp32a LRR domain, and U2A¢ (C) [20]. The coordi-
nates were first superimposed using the
DALI server, and then
displaced.
C. de Chiara et al. Study of the interactions of the Anp32a LRR module
FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS 2551
U2A¢ (residues 13–26) provides further interactions by
wrapping around the other molecule on one side.
There is also a good charge complementarity, as the
concave surface of the U2A¢ LRR is negatively

charged, whereas the U2B¢¢ helix, which is neutral
overall, contains at least one positively charged residue
(Arg28), which protrudes out into the solvent and is
Fig. 3. Relaxation parameters and RDC values along the sequence
of the Anp32a LRR domain. The data were recorded at 27 °C and
800 MHz.
A
B
Fig. 4. Modeling the interactions of the Anp32a LRR domain. (A)
Structure of the Anp32a LRR domain in a complex with the C sub-
unit of PP2A as modeled by comparison with the U2A¢–U2B¢¢
complex. The other two subunits shield most of the surface of
PP2A–C. (B) Structure of the U2A¢–U2B¢¢ complex [20].
Study of the interactions of the Anp32a LRR module C. de Chiara et al.
2552 FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS
able to form a salt bridge with Glu92 of U2A¢. Other
contacts will contribute with hydrophobic interactions
or intermolecular hydrogen bonds, which are likely to
be responsible for the specificity of recognition, which
seems to be tuned to the specific system. Accordingly,
the U1A protein, which is closely related to U2B¢¢,
does not form a stable complex with the U2A¢ LRR,
whereas replacement of Asp24 and Lys28 with the
homologous Glu and Arg of U2B¢¢ [20] is sufficient to
re-establish formation of the complex [31].
We modeled, as a paradigmatic and particularly inter-
esting example, a complex between PP2A and the
Anp32a LRR. The structure of PP2A has recently been
determined [13,14]. It consists of a heterotrimeric com-
plex formed by the scaffolding subunit A, the regulatory

subunit B¢⁄B56 ⁄ PR61, and the catalytic domain C.
Interaction with Anp32 has been shown to involve the
catalytic subunit [15,32] and to inhibit its catalytic
activity, both in the absence and in the presence of the
scaffold subunit A and the regulatory subunit B, with
apparent K
i
in the low nanomolar range [4]. This implies
that the interaction involves an exposed region of
PP2A-C, without appreciable contributions from the
other two subunits. Anp32 is also known to inhibit
PP2A in a noncompetitive manner, i.e. without binding
to the active site of the enzyme [4]. Finally, antibodies
recognizing the fourth LRR of Anp32e ⁄ Cpd1 (resi-
dues 87–101) are known to block the inhibitory PP2A
activity of Anp32e in protein extracts [7]. Taken
together, these findings limit the region of interaction to
the only exposed surface of PP2A-C that contains a
semiexposed helix (residues 222–232).
The model of an Anp32 LRR–PP2A complex, built
using complex U2A¢–U2B¢¢ as a template, shows that,
by analogy with this structure, helix 222-232 of PP2A-
C protrudes out enough to fit well into the groove
formed by the concave surface of the LRR domain.
Stabilizing interactions could form between His230 of
PP2A-C and Asp119 and Asn94 of Anp32a. A salt
bridge could form between Glu226 of PP2A-C and
Lys67, Lys68 and Lys91.
Testing the interaction with Atx1 experimentally
Interaction between the Anp32a LRR domain and the

Atx1 AXH domain was tested experimentally by
NMR chemical shift perturbation, with the aim of
mapping the surface of interaction between the two
proteins. This method, which relies on the effect that
binding of a molecule has on the electron distribution
of another, causing a perturbation of its NMR spec-
trum, is routinely used to detect interactions and map
them on the structures of the individual components.
We titrated the LRR domain with the AXH domain
since this region had been proposed to be essential for
the interaction on the basis of deletion mutants [18].
When the effects were mapped onto the Anp32 surface
(Fig. 5), they all clustered around the concave surface.
However, even at high Atx1 AXH ⁄ Anp32a LRR ratios
(3.5 : 1 and low ionic strength), we observed only min-
imal perturbations of the Anp32 LRR domain spec-
trum (i.e.: <0.05 ppm in the proton dimension), which
were absent in spectra recorded at a higher ionic
strength (150 mm NaCl). Likewise, when we titrated
the Atx1 AXH domain with the Anp32a LRR domain,
we observed only two very small effects.
The interaction was independently probed by fluo-
rescence spectroscopy, exploiting the intrinsic emission
at 327 nm of the only Trp residue present in Atx1
(Trp658; Anp32a does not contain Trp residues) after
sample excitation at 295 nm. During titration, fluores-
cence quenching was observed along with a 4 nm blue
shift of the k
max
of emission (from 327 to 323 nm),

suggesting a decrease in the Trp solvent exposure con-
sequent to interaction (data not shown). However, the
decrease in fluorescence intensity was far from reach-
ing a plateau even at the highest Anp32a ⁄ Atx1 ratio
tested (60 : 1).
Fig. 5. Probing the interaction between the Anp32a LRR domain
and the AXH domain of Atx1 by chemical shift perturbation. Super-
imposition of the HSQC spectra of a 0.2 m
M solution of
15
N-labeled
Anp32a LRR domain in 20 m
M Tris (pH 7.0) and 2 mM TCEP,
recorded at 600 MHz and 27 °C in the absence (blue) and in the
presence (red) of a three-fold excess of unlabeled Atx1 AXH
domain.
C. de Chiara et al. Study of the interactions of the Anp32a LRR module
FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS 2553
This evidence indicates that interaction between the
two domains is very weak, i.e. with binding constants
in the millimolar range. Although such binding is defi-
nitely too weak to be significant, it is certainly possible
that, in vivo, the interaction is enhanced either by other
regions of the two molecules or by post-translational
modifications that are absent in our assays.
Identification of new potential partners of the
Anp32 LRR domain
To identify new partners specific for the Anp32a LRR
domain, we used a construct spanning the same region
studied by structural techniques (residues 1–164) as a

bait in a two-hybrid screening assay. This is at vari-
ance with previous studies, which were all carried out
on the full-length protein, thus inferring the role of the
LRR domain only indirectly. By screening of a human
brain cDNA library (Clonetech, Mountain View, CA,
USA) for a total of approximately 5 million clones, we
found about 600 potential positives [i.e. hits that were
positive both for quadruple-dropout media and for
5-bromo-4-chloroindol-3-yl b-d-galactoside (Gal-X)
overlay assays]. Nearly 200 of these positives were
sequenced. Among these, we identified 29 clones of the
C-terminus of the microtubule-associated protein
(MAP) Clip 170 ⁄ Restin, a microtubule plus-end track-
ing protein (+TIP), which associates with and regu-
lates the dynamic properties of microtubules and of
other MAPs [33] (Fig. 6).
We tested the interaction further by expressing the
full-length proteins in mammalian cells. In transfected
COS cells, Anp32a was predominantly nuclear, with a
limited number of cells showing extranuclear staining
(Fig. 7A). In contrast, and as expected, Clip 170 was
excluded from the nucleus and localized to the micro-
tubule network. Partial colocalization of Clip 170 and
Anp32a was observed in the microtubules of cells
coexpressing these proteins and showing extranuclear
staining of Anp32a (Fig. 7A, merged image).
To further validate the interaction, we carried out
coimmunoprecipitation experiments to test the ability
of the endogenous proteins to associate. HeLa cell
lysates were immunoprecipitated with antibodies to

Anp32a or with antibodies to histone H3 as a negative
control. The proteins from immunoprecipitation com-
plexes were subjected to western blot analysis using
antibodies to Clip 170. Clip 170 was associated with
the complex pulled down by antibodies to Anp32a
but not with the one pulled down by antibodies to
histone H3 (Fig. 7B,C).
Discussion
Here, we have explored the interaction properties of
the LRR domain of Anp32, a family of LRR proteins
potentially implicated in several important cellular
pathways. Two particularly interesting interactions
have been described, with the PP2A phosphatase and
with Atx1, two proteins of high medical importance.
We first determined the domain boundaries of the
domain by solving the solution structure at high reso-
lution of a fragment spanning the whole conserved
region up to some highly acidic repeats containing EA-
EEE motifs. We show that the domain contains a
compact and rigid fold with five LRRs and a
C-capping motif. The structural information was used
to model the interaction with PP2A, which is known
to be mainly mediated by the PP2A-C subunit. We
suggest that, by analogy with the mode of recognition
of U2B¢¢ by U2A¢, which has the highest structural simi-
larity with the Anp32a LRR domain, the interaction
+
+
-
-

A
B
BD-Lanp.NT
BD-Lanp.NT
AD-Clip.CT
AD-Clip.CT
AD-Clip.NT
+
+
–
–
BD
––
Bait
Prey
Growth on QD plates
X-Gal overlay
EMKKRESKFIKDADEEKASLQKSISITSALLTEKDAELEKLRNEVTVLRGENASAKSLHSVVQTLESDK
VKLELKVKNLELQLKENKRQLSSSSGNTDTQADEDERAQESQIDFLNSVIVDLQRKNQDLKMKVEM
MSEAALNGNGDDLNNYDSDDQEKQSKKKPRLFCDICDCFDLHDTEDCPTQAQMSEDPPHSTHHGS
RGEERPYCEICEMFGHWATNCNDDETF
Fig. 6. Interaction of Clip 170 with Anp32a
in a yeast two-hybrid system. (A) The N-ter-
minus of Anp32a fused to the Gal4 DNA-
binding domain (BD-Lanp.NT) interacts with
the C-terminus of Clip 170 (AD-Clip.CT)
fused to the Gal4 DNA activation domain as
indicated by growth on quadruple-dropout
(QD) plates and Gal-X overlay assays. There

was no growth on QD plates when either
the N-terminus of Clip 170 (residues 1–
1164, BD-Clip.NT) or the Gal4 DNA-binding
domain was used as prey. (B) Amino acid
sequence of the region of Clip 170 interact-
ing with Anp32a.
Study of the interactions of the Anp32a LRR module C. de Chiara et al.
2554 FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS
involves the helix-spanning residues 222–230 of PP2A-
C [13,14]. This region is the only element of PP2A-C
protruding out from the PP2A trimer, and its size and
shape mean that it could easily fit into the complemen-
tary concave surface of the Anp32a LRR domain.
We tested binding to the AXH domain of Atx1
experimentally by chemical shift perturbation assays.
We observed only very minor effects, which are com-
patible, at the very best, with millimolar affinities.
The effects could be observed only at low ionic
strength, suggesting that the interaction is mainly of
an electrostatic nature and is nonspecific. Would our
results shed doubts on an interaction originally
observed by two-hybrid screening? On the one hand,
it is interesting to note that none of the high-through-
put studies of the Atx1 interactome has reported any
evidence for this interaction [34,35]. On the other
hand, however, very recent data provide the first evi-
dence of a functional link between Anp32a and Atx1,
showing that Atx1 relieves the transcriptional repres-
sion induced by Anp32a in complex with E4F [36].
As addition of exogenous Anp32a restores repression,

it was suggested that Atx1 sequesters Anp32a, releas-
ing its interaction with E4F. Our evidence may there-
fore indicate that either a third component (which
could be another region of one or both proteins or
another molecule) or a post-translational modification
is needed to give appreciable affinities. The second
possibility seems currently most likely: both Atx1 and
Anp32a are known to be natively phosphorylated,
and phosphorylation has been shown to modulate
some of their functions [1,3,37]. Atx1 contains two
phosphorylation sites, both outside the AXH domain,
one of which (Ser776) is located in the C-terminal
region of the protein and is known to modulate the
interaction with 14-3-3 [37,38]. Two in vivo CK2
phosphorylation sites (Ser158 and Ser204) have also
been identified in Anp32a [26]. Phosphorylation of
Ser158, which is immediately downstream of the
LRR domain, could, for instance, induce a conforma-
tional change of the adjacent region, which could be
required for Atx1 binding.
Finally, we used yeast two-hybrid screening to iden-
tify new partners of Anp32a. To our knowledge, ours
is the first study carried out using, for library screen-
ing, the LRR domain only, i.e. excluding the acidic
C-terminus, which, being highly charged, could pro-
duce false positives. We observed an interaction
between the Anp32a LRR domain and the microtubule
+TIP Clip 170. This protein is known to associate
A
BC

Fig. 7. Anp32a and Clip 170 associate with each other in HeLa and transfected COS cells. (A) Colocalization of Clip 170 and Anp32a in COS
cells that were transfected with a plasmid vector carrying V5-tagged Clip 170 and c-Myc-tagged Anp32a. Cells were analyzed by confocal
microscopy. Clip 170 was localized in the microtubule network (green), and Anp32a (red) was predominantly nuclear, with some cells show-
ing localization in the microtubules. The merged image shows colocalization of the proteins in the microtubules. (B) Expression of endoge-
nous proteins in HeLa cells. HeLa cells were lysed in RIPA buffer, and input controls and immunoprecipitated samples were probed with
the antibodies shown. (C). Interaction of endogenous Clip 170 and Anp32a in HeLa cells. HeLa cells were lysed in RIPA buffer and immuno-
precipitated as above with antibodies to histone H3 or antibodies to Anp32a. Proteins were transferred onto a poly(vinylidene difluoride)
membrane and probed with antibodies to Clip 170.
C. de Chiara et al. Study of the interactions of the Anp32a LRR module
FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS 2555
with microtubules and with other MAPs, and to regu-
late the dynamic properties of microtubules [33]. Iden-
tification of this new potential partner is particularly
interesting, because Anp32a has already been reported
to be involved in microtubule dynamics via its interac-
tion with several members of the family of MAPs, i.e.
MAP1B, MAP2, and MAP4 [39–41]. The interaction
with MAP1B was suggested to modulate the effects of
MAP1B in neurite extension [41]. Microtubule +TIPs
have also been shown to be involved in modulating
neuronal growth cones, the motile tips of growing
axons [42,43]. Interaction of Clip 170 with micro-
tubules has been suggested to be influenced by
phosphorylation, as phosphorylation by a rampamy-
cin-sensitive kinase (fluorescence recovery after photo-
bleaching; FRAP) increases the interaction of Clip 170
with microtubules [44]. Interestingly, in our coimmu-
noprecipitation experiments, the Clip 170 band
appeared to be more intense when the cell lysates
incorporated a cocktail of phosphatase inhibitors, sug-

gesting that the association may be modulated by
phosphorylation events (not shown).
Like Anp32, which is linked to the SCA1 pathology
[16], Clip 170 is also known to be associated with
human disease. The protein is overexpressed in Hodg-
kin’s disease and anaplastic large cell lymphoma
[45,46]. Clip 170 has also been shown to interact with
the Lis1 protein, whose mutation causes type I lissen-
cephaly, a severe brain developmental disease [47].
Therefore, our results point out to an important role
of Anp32 proteins in human pathologies and encour-
age further studies to clarify the complete interactome
of this protein.
Experimental procedures
Protein sample preparation
The LRR domain of Anp32a from Mus musculus (resi-
dues 1–164) was produced using an ampicillin-resistant glu-
tathione S-transferase (GST)-3C expression vector with a
human rhinovirus 3C protease recognition site. This con-
struct resulted in the addition of five non-native residues
(GPLGS) at the N-terminus of the protein. Isotopically
15
N-labeled and
13
C ⁄
15
N-labeled samples were overexpres-
sed in the Escherichia coli host strain BL21 (DE3) grown
on a minimal medium containing [
15

N]ammonium sulfate
and [
13
C]glucose as the sole sources of nitrogen and carbon
respectively. The cells were grown at 37 °C until an attenu-
ance (D) at 600 nm of 0.5 was reached, and then cooled
to 18 °C, induced with isopropyl thio-b-d-galactoside
(0.5 mm), and harvested after overnight expression. A stan-
dard purification protocol was performed, using Pharmacia
GST–Sepharose resin (GE Healthcare). Cleavage of the
GST tag was achieved overnight at room temperature using
the PreScission protease (GE Healthcare). The protein was
further purified by HPLC size exclusion chromatography,
using a prepacked HiLoad 16 ⁄ 60 Superdex 75 prep grade
column (Pharmacia). The concentration of the NMR sam-
ple used for structural studies was typically in the range
0.3–0.7 mm, in a buffer containing 10 mm Tris ⁄ HCl and
2mm Tris(2-carboxyethyl)phosphine (TCEP) at pH 7.0 in
90% H
2
O ⁄ 10% D
2
O. All the NMR experiments were per-
formed at 27 °C on Bruker Advance and Varian Inova
spectrometers, both equipped with cryoprobes and operat-
ing at 14.1 and 18.8 T, respectively, and on a Varian Inova
spectrometer operating at 14.1 T. Samples of the Atx1
AXH domain (residues 567–689 and 567–694) were pro-
duced as previously described [18].
Experimental restraints

Resonance assignment of the LRR domain was performed
as previously described [21]. Interproton distance restraints
were derived from NOESY
15
N HSQC and NOESY
13
C HSQC spectra acquired at 27 °C with mixing times of
100 ms on a Varian Inova spectrometer operating at
800 MHz
1
H frequency. A set of 89 backbone / and u
dihedral angles was obtained using the backbone torsion
angle prediction package talos [48]. Amide protection
was inferred from deuterium exchange measurements per-
formed at 27 °C on a freeze-dried
15
N-labeled sample
redissolved in a Tris ⁄ HCl-buffered (pH 7.0) D
2
O solution
and started immediately after redissolving the protein. The
intensity decay of the NH signals extracted from a series
of 40
1
H–
15
N HSQCs of 35 min each allowed calculation
of the exchange rates. Twenty slowly exchanging protons
were identified as having an exchange time longer than
3 h. Among these, a hydrogen bond restraint was added if

a hydrogen bond was consistently observed in at least
50% of the structures inspected at an advanced stage of
the refinement.
1
D
NH
RDCs were measured at 27 °C,
aligning the protein in 5% n-dodecyl-penta(ethylene gly-
col) ⁄ n-hexanol (r = 0.92) using a buffer composed of
20 mm Tris ⁄ HCl, 2 mm TCEP and 0.02% NaN
3
at
pH 7.0. The liquid crystalline medium gave a stable quad-
rupolar splitting of the D
2
O signal of 21 Hz. The final
concentration of the protein in this medium was
 0.37 mm.92
1
J
NH
splittings were obtained from a
J-modulated
15
N–
1
H HSQC spectrum [49] for NH vectors
with a heteronuclear NOE value higher than 0.75 and
used for the purpose of structure validation using the
program module [50]. The rmsd in hertz from RDC

restraints (observed – calculated from structure generated
without using RDCs) is 0.620 ± 0.035.
T
1
,T
2
and heteronuclear NOE measurements were per-
formed at 27 °C and 800 MHz, using adapted standard
Study of the interactions of the Anp32a LRR module C. de Chiara et al.
2556 FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS
pulse sequences. The T
1
⁄ T
2
ratios of residues not undergo-
ing large amplitude motions or exchange were used to esti-
mate the correlation time (s
c
), assuming the model-free
approach [28]. Residues with T
1
and T
2
values that differed
by more than one standard deviation from the mean were
excluded from the s
c
calculation.
Structure calculation for Anp32a
Structure calculations were performed using the aria pro-

gram (version 1.2) [51]. A typical run consisted of nine iter-
ations. At each iteration, 20 structures were calculated by
simulated annealing using the standard cns protocol [52]
with numbers of steps equal to 15 000 and 12 000 in the
first and second cooling stages of the annealing, respec-
tively. Floating assignment for prochiral groups and correc-
tion for spin diffusion during iterative NOE assignment
were applied as previously described [53,54]. At the end of
each iteration, the best seven structures in terms of lowest
global energy were selected and used for assignment of
additional NOEs during the following iteration. In the final
aria run, the number of structures generated in iteration 8
was increased to 100, and after refinement by molecular
dynamics simulation in water of the 50 lowest-energy struc-
tures [55], the 10 lowest-energy structures were selected as
representative of the Anp32a LRR domain structure and
used for statistical analysis. In the final iteration, 3774
unambiguous and 1377 ambiguous NOEs were assigned.
Among the 5151 total NOEs, 2021 were intraresidue, 1075
sequential, 663 medium range, and 1392 long range. Struc-
ture quality was evaluated using the programs procheck
[56] and whatif [57]. The coordinates are deposited with
the Protein Data Bank (accession code 2jqd).
Comparative modeling
The structure of an Anp32a–PP2A complex was modeled
on the U2A¢–U2B¢¢ coordinates (1a9n) [20]. The available
information strongly indicates that the interaction is domi-
nated by the C subunit of PP2A. Of this, the main region
that protrudes out into solution and is not protected by
interactions with the other two subunits comprises

helix 222–232. Assuming a similar modality of interaction,
we superimposed this region on helix 1 of U2B¢¢ (resi-
dues 24–34). The resulting complex did not involve major
steric clashes except with the flexible C-terminus of Anp32a.
The structure was energy minimized by the gromacs pack-
age [58] using the gromos96 force field [59] to relieve possi-
ble structural strain.
Atx1 interactions
Interaction of Anp32a with the Atx1 AXH domain was
probed both by NMR spectroscopy and by fluorescence
spectroscopy. Two different constructs of the Atx1 AXH
domain with different C-terminal boundaries (residues 567–
689 or residues 567–694) were used. Typically, 0.2–0.3 mm
solutions of the
15
N-labeled Anp32a LRR domain in
20 mm Tris (pH 7.0) and 2 mm TCEP were used for the
NMR experiments. They were titrated with stepwise addi-
tions of concentrated stock solutions of unlabeled Atx1
AXH domain up to a two to threefold excess of this. The
inverse titration using labeled Atx1 AXH domain and unla-
beled LRR domain was also probed. The experiments were
carried out at 27 °C, both at low ionic strength to enhance
even weak electrostatic interactions, and at physiological
ionic strength (150 mm NaCl).
Fluorescence measurements were performed on a SPEX
Fluoromaxspectrometer, by exciting at 295 nm (slit width
0.4 nm) a 10 lm sample of Atx1 AXH domain in 20 mm
Tris (pH 7.0) and recording the emission intensity from 300
to 450 nm (slit width 1.5 nm). Titration was carried out by

stepwise additions of a 0.87 mm stock solution of Anp32a
LRR domain up to a 60 : 1 ratio. The data were evaluated
using the origin program package (Micro-Cal Software,
Bletchley, UK).
Yeast two-hybrid analysis
The DNA fragment encoding the murine Anp32a N-termi-
nus (1–164 amino acids) was cloned into the pGBKT7 vec-
tor (Clontech, Mountain View, CA, USA) for expression as
a Gal4 DNA-binding domain fusion protein. This bait was
transformed into an AH 109 yeast strain and used to screen
a human brain two-hybrid cDNA library from Clonetech
as previously described [60]. DNAs recovered from clones
selected by growth in quadruple-dropout media and Gal-X
overlay assays were sequenced and compared with known
sequences.
Confocal microscopy
cDNAs encoding full-length Anp32a and Clip 170 (Gene-
Service, IMAGE 3592614) were cloned into the
pBudCE4.1 vector (Invitrogen, Paisley, UK). The immu-
nofluorescence assay was carried out essentially as
described previously [61]. Briefly, COS cells were grown
overnight in chamber slides and transfected with
pBudCE4.1 vector expressing V5-tagged Clip 170 and c-
Myc-tagged Anp32a. Forty-eight hours after transfection,
cells were fixed using 4.0% paraformaldehyde, permeabi-
lized with 0.2% Triton X-100 ⁄ NaCl ⁄ P
i
and probed with
fluorescein isothiocyanate-conjugated antibodies to V5
(Invitrogen) and Cy3-conjugated antibodies to c-Myc

(Sigma, Poole, UK) for 1 h at room temperature. After
being washed with NaCl ⁄ P
i
, slides were mounted using
Citifluor (Agar Scientific) before analysis by confocal
microscopy. Cells were visualized under a Leica laser
C. de Chiara et al. Study of the interactions of the Anp32a LRR module
FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS 2557
scanning confocal microscope (TCS-SP1) equipped with a
DM-RXE microscope and an argon–krypton laser.
Images were acquired as single 0.2 lm transcellular opti-
cal sections and averaged over 20 scans ⁄ frame. Images
were acquired sequentially, and appropriate emission filter
settings and controls were included to minimize bleed-
through effects. Images were merged using the image j
program (NIH, Bethesda). Merged images show the
details observed in a single 0.2 lm optical section.
Immunoprecipitation
HeLa cells were grown to confluency and were subsequently
lysed using RIPA buffer (50 mm Tris ⁄ HCl, pH 8.0, 150 mm
NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1.0% NP-40,
supplemented with complete protease inhibitor cocktail tab-
lets (Roche, Basel, Switzerland). Cleared lysates were incu-
bated with protein A and protein G agarose beads (Sigma)
for 30 min, spun down to remove the beads, and incubated
overnight at 4 °C with monoclonal antibodies to Clip 170
or polyclonal antibodies to Anp32a (Santacruz Biotech,
Santa Cruz, CA, USA), or with polyclonal antibodies to
histone H3 (Calbiochem), along with fresh protein A ⁄ G
agarose beads. Beads were spun down and washed three

times with RIPA buffer before being resuspended in
SDS ⁄ PAGE sample buffer. Samples and input controls
were analyzed by PAGE and western blotting using mono-
clonal antibodies to Clip 170, histone H3 or Anp32a.
Acknowledgements
We thank Drs N. Q. McDonald and B. O’Hara (Bir-
beck College, London) for providing the Lanp clone,
which was produced from a cDNA originally provided
by Dr A. Matilla (ICH, London), Filippo Prischi for
help with the gromacs software, and Dr L Masino for
helpful discussions. The project is under the Eurosca
consortium.
References
1 Matilla A & Radrizzani M (2005) The Anp32 family of
proteins containing leucine-rich repeats. Cerebellum 4,
7–18.
2 Malek SN, Katumuluwa AI & Pasternack GR (1990)
Identification and preliminary characterization of two
related proliferation-associated nuclear phosphopro-
teins. J Biol Chem 265, 13400–13409.
3 Santa-Coloma TA (2003) Anp32e (Cpd1) and related
protein phosphatase 2 inhibitors. Cerebellum 2, 310–320.
4 Li M, Guo H & Damuni Z (1995) Purification and
characterization of two potent heat-stable protein inhib-
itors of protein phosphatase 2A from bovine kidney.
Biochemistry 34, 1988–1996.
5 Li M, Makkinje A & Damuni Z (1996a) The myeloid
leukemia-associated protein is a potent inhibitor of pro-
tein phosphatase 2A. J Biol Chem 271, 11059–11062.
6 Li M, Makkinje A & Damuni Z (1996b) Molecular

identification of I1PP2A, a novel potent heat-stable
inhibitor protein of protein phosphatase 2A. Biochemis-
try 35, 6998–7002.
7 Radrizzani M, Vila-Ortiz G, Cafferata EG, Di Tella
MC, Gonzalez-Guerrico A, Perandones C, Pivetta OH,
Carminatti H, Idoyaga Vargas VP & Santa-Coloma TA
(2001) Differential expression of CPD1 during postnatal
development in the mouse cerebellum. Brain Res 907,
162–174.
8 Costanzo RV, Vila
´
-Ortı
´
z GJ, Perandones C, Carminatti
H, Matilla A & Radrizzani M (2006) Anp32e ⁄ Cpd1
regulates protein phosphatase 2A activity at synapses
during synaptogenesis. Eur J Neurosci 23, 309–324.
9 Mumby M (2007) The 3D structure of protein phospha-
tase 2A: new insights into a ubiquitous regulator of cell
signaling. ACS Chem Biol 2, 99–103.
10 Mumby M (2007b) PP2A: unveiling a reluctant tumor
suppressor. Cell 130, 21–24.
11 Janssens V & Goris J (2001) Protein phosphatase 2A: a
highly regulated family of serine ⁄ threonine phosphata-
ses implicated in cell growth and signalling. Biochem J
353, 417–439.
12 Janssens V, Goris J & Van Hoof C (2005) PP2A: the
expected tumor suppressor. Curr Opin Genet Dev 15,
34–41.
13 Xu Y, Xing Y, Chen Y, Chao Y, Lin Z, Fan E, Yu

JW, Strack S, Jeoffrey PD & Shi Y (2006) Structure of
the protein phosphatase 2A holoenzyme. Cell 127,
1239–1251.
14 Cho US & Xu W (2007) Crystal structure of a protein
phosphatase 2A heterotrimeric holoenzyme. Nature 445,
53–57.
15 Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck
FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr
A, Koeppen S et al. (2005) A human protein–protein
interaction network: a resource for annotating the pro-
teome. Cell 122, 957–968.
16 Matilla A, Koshy BT, Cummings CJ, Isobe T, Orr HT
& Zoghbi HY (1997) The cerebellar leucine-rich acidic
nuclear protein interacts with ATX1. Nature 389, 974–
978.
17 Masino L & Pastore A (2002) Glutamine repeats: struc-
tural hypotheses and diseases. Biochem Soc Trans 29,
41–60.
18 de Chiara C, Giannini C, Adinolfi S, de Boer J, Guida S,
Ramos A, Jodice C, Kioussis D & Pastore A (2003) The
AXH module: an independently folded domain common
to ataxin-1 and HBP1. FEBS Lett 551, 107–112.
19 Huyton T & Wolberger C (2007) The crystal structure
of the tumour suppressor protein pp32 (Anp32a):
Study of the interactions of the Anp32a LRR module C. de Chiara et al.
2558 FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS
structural insights into Anp32 family of proteins. Prot
Sci 16, 1308–1315.
20 Price SR, Evans PR & Nagai K (1998) Crystal structure
of the spliceosomal U2B¢¢–U2A¢ protein complex bound

to a fragment of U2 small nuclear RNA. Nature 394,
645–650.
21 de Chiara C, Kelly G, Frenkiel TA & Pastore A (2007)
NMR assignment of the leucine-rich repeat domain of
LANP ⁄ Anp32a. J Biomol NMR 38, 177.
22 Holm L & Sander C (1996) Mapping the protein uni-
verse. Science 273, 595–603.
23 Kobe B & Kajava AV (2001) The leucine-rich repeat as
a protein recognition motif. Curr Opin Struct Biol 11,
725–732.
24 Seo SB, Macfarlan T, McNamara P, Hong R, Mukai
Y, Heo S & Chakravarti D (2002) Regulation of his-
tone acetylation and transcription by nuclear protein
pp32, a subunit of the INHAT complex. J Biol Chem
277, 14005–14010.
25 Brody JR, Kadkol SS, Mahmoud MA, Rebel JM &
Pasternack GR (1999) Identification of sequences
required for inhibition of oncogene-mediated transfor-
mation by pp32. J Biol Chem 274, 20053–20055.
26 Hong R, Macfarlan T, Kutney SN, Seo SB, Mukai Y,
Yelin F, Pasternack GR & Chakravarti D (2004) The
identification of phosphorylation sites of pp32 and bio-
chemical purification of a cellular pp32-kinase. Biochem-
istry 43, 10157–10165.
27 Bairoch A (1992) PROSITE: a dictionary of sites and
patterns in proteins. Nucleic Acids Res 20(Suppl.),
2013–2018.
28 Lipari G & Szabo A (1982) Model-free approach to the
interpretation of nuclear magnetic resonance relaxation
in macromolecules. 1. Theory and range of validity.

J Am Chem Soc 104, 4546–4559.
29 Maciejewski M, Liu D, Prasad R, Wilson S & Mullen
G (2000) Backbone dynamics and refined solution struc-
ture of the N-terminal domain of DNA polymerase
beta. Correlation with DNA binding and dRP lyase
activity. J Mol Biol 296, 229–253.
30 Sanders CR, Hare BJ, Howard KP & Prestegard JH
(1993) Magnetically-oriented phospholipid micelles as a
tool for the study of membrane-associated molecules.
Prog Nucl Magn Reson Spectrosc 26, 421–444.
31 Scherly D, Dathan NA, Boelens W, van Venrooij WJ &
Mattaj IW (1990) The U2B¢¢ RNP motif as a site of
protein–protein interaction. EMBO J 9, 3675–3681.
32 Chen S, Li B, Grundke-Iqbal I & Iqbal K (2008) I
1
PP2A
affects Tau phosphorylation via association with the
catalytic subunit of protein phosphatase 2A. J Biol
Chem doi: 10.1074/jbc.M709852200.
33 Galjart N (2005) CLIPs and CLASPs and cellular
dynamics. Nat Rev Mol Cell Biol 6, 487–498.
34 Lim J, Hao T, Shaw C, Patel AJ, Szabo G, Rual JF,
Fisk CJ, Li N, Smolyar A, Hill DE et al. (2006) A
protein–protein interaction network for human inher-
ited ataxias and disorders of Purkinje cell degeneration.
Cell 125, 801–814.
35 Gould R, Hubank M, Hunt A, Holton J, Menon RP,
Revesz T, Pandolfo M & Matilla-Duenas A (2007)
Down-regulation of the dopamine receptor D2 in mice
lacking ataxin 1. Hum Mol Genet 16, 2122–2134.

36 Cvetanovic M, Rooney RJ, Garcia JJ, Toporovskaya
N, Zoghbi HY & Opal P (2007) The role of LANP and
ataxin 1 in E4F-mediated transcriptional repression.
EMBO Rep 8, 671–677.
37 Emamian ES, Kaytor MD, Duvick LA, Zu T, Tousey
SK, Zoghbi HY, Clark HB & Orr HT (2003) Serine 776
of ataxin-1 is critical for polyglutamine-induced disease
in SCA1 transgenic mice. Neuron 38, 375–387.
38 Vierra-Green CA, Orr HT, Zoghbi HY & Ferrington
DA (2005) Identification of a novel phosphorylation site
in ataxin-1. Biochim Biophys Acta 1744, 11–18.
39 Ulitzur N, Humbert M & Pfeffer SR (1997) Mapmodu-
lin: a possible modulator of the interaction of microtu-
bule-associated proteins with microtubules. Proc Natl
Acad Sci USA 94 , 5084–5089.
40 Ulitzur N, Rancano C & Pfeffer SR (1997) Biochemical
characterization of mapmodulin, a protein that binds
microtubule-associated proteins. J Biol Chem 272,
30577–30582.
41 Opal P, Garcia JJ, Propst F, Matilla A, Orr HT &
Zoghbi HY (2003) Mapmodulin ⁄ leucine-rich acidic
nuclear protein binds the light chain of microtubule-
associated protein 1B and modulates neuritogenesis.
J Biol Chem 278, 34691–34699.
42 Lee H, Engel U, Rusch J, Scherrer S, Sheard K & Van
Vactor D (2004) The microtubule plus end tracking
protein Orbit ⁄ MAST ⁄ CLASP acts downstream of the
tyrosine kinase Abl in mediating axon guidance. Neuron
42, 913–926.
43 Zhou FQ, Zhou J, Dedhar S, Wu YH & Snider WD

(2004) NGF-induced axon growth is mediated by local-
ized inactivation of GSK-3beta and functions of the
microtubule plus end binding protein APC. Neuron 42,
897–912.
44 Choi JH, Bertram PG, Drenan R, Carvalho J, Zhou
HH & Zheng XF (2002) The FKBP12-rapamycin-asso-
ciated protein (FRAP) is a CLIP-170 kinase. EMBO
Rep 3, 988–994.
45 Bilbe G, Delabie J, Bruggen J, Richener H, Asselbergs
FA, Cerletti N, Sorg C, Odink K, Tarcsay L, Wiesen-
danger W et al. (1992) Restin: a novel intermediate
filament-associated protein highly expressed in the
Reed–Sternberg cells of Hodgkin’s disease. EMBO J
11, 2103–2113.
46 Delabie J, Shipman R, Bruggen J, De Strooper B, van
Leuven F, Tarcsay L, Cerletti N, Odink K, Diehl V,
Bilbe G et al. (1992) Expression of the novel intermedi-
ate filament-associated protein restin in Hodgkin’s dis-
C. de Chiara et al. Study of the interactions of the Anp32a LRR module
FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS 2559
ease and anaplastic large-cell lymphoma. Blood 80,
2891–2896.
47 Coquelle FM, Caspi M, Cordelieres FP, Dompierre JP,
Dujardin DL, Koifman C, Martin P, Hoogenraad CC,
Akhmanova A, Galjart N et al. (2002) LIS1, CLIP-
170’s key to the dynein ⁄ dynactin pathway. Mol Cell
Biol 22, 3089–3102.
48 Cornilescu G, Delaglio F & Bax A (1999) Protein back-
bone angle restraints from searching a database for
chemical shift and sequence homology. J Biomol NMR

13, 289–302.
49 Ottinger M, Delaglio F & Bax A (1998) Measurement
of J and dipolar couplings from simplified two-dimen-
sional NMR spectra. J Magn Res 131, 373–378.
50 Dosset P, Hus JC, Marion D & Blackledge M (2001) A
novel interactive tool for rigid-body modeling of multi-
domain macromolecules using residual dipolar cou-
plings. J Biomol NMR 20, 223–231.
51 Linge JP, O’Donoghue SI & Nilges M (2001)
Automated assignment of ambiguous nuclear
Overhauser effects with ARIA. Methods Enzymol 339,
71–90.
52 Brunger AT, Adams PD, Clore GM, DeLano WL,
Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski
J, Nilges M, Pannu NS et al. (1998) Cystallography
and NMR system: a new software suite for macromo-
lecular structure determination. Acta Crystallogr 54,
905–921.
53 Folmer RH, Hilbers CW, Konings RN & Nilges M
(1997) Floating stereospecific assignment revisited:
application to an 18 kDa protein and comparison with
J-coupling data. J Biomol NMR 9, 245–258.
54 Linge JP, Habeck M, Rieping W & Nilges M (2004)
Correction of spin diffusion during iterative automated
NOE assignment. J Magn Res 167, 334–342.
55 Linge JP, Williams MA, Spronk CA, Bonvin AM &
Nilges M (2003) Refinement of protein structures in
explicit solvent. Proteins 50, 496–506.
56 Laskowski RA, Rullman JA, MacArthur MW, Kaptein
R & Thornton JM (1996) AQUA and PROCHECK-

NMR: programs for checking the quality of protein
structures solved by NMR. J Biomol NMR 8, 477–486.
57 Vriend G (1990) WHAT IF: a molecular modelling and
drug design program. J Mol Graph 8, 52–56.
58 Berendsen HJC, van der Spoel CD & van Drunen R
(1995) GROMACS: a message-passing parallel molecu-
lar dynamics implementation. Comp Phys Commun 91,
43–56.
59 Van Gunsteren WF, Billeter S, Eising A, Hunenberger
PH, Kruger P, Mark AE, Scott WRP & Tironi IG
(1996) Biomolecular Simulations: the GROMOS96 Man-
ual and User Guide. VdF: Hoshshulverlag AG an der
ETH Zurich and BIOMOS b.v, Zurich, Gronigen.
60 Menon RP, Gibson TJ & Pastore A (2004) The C ter-
minus of fragile X mental retardation protein interacts
with the multi-domain Ran-binding protein in the
microtubule-organising centre. J Mol Biol 343, 43–53.
61 Menon RP & Pastore A (2006) Expansion of amino
acid homo-sequences in proteins: insights into the role
of amino acid homo-polymers and of the protein con-
text in aggregation. Cell Mol Life Sci 63, 1677–1685.
62 Thomas DJ & Pastore A (2004) WHEATSHEAF – an
algorithm to average protein structure ensembles. Acta
Crystallogr
61, 112–116.
Study of the interactions of the Anp32a LRR module C. de Chiara et al.
2560 FEBS Journal 275 (2008) 2548–2560 ª 2008 The Authors Journal compilation ª 2008 FEBS