Structural bases for recognition of Anp32/LANP proteins

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  • Structural bases for recognition of Anp32 LANP proteinsCesira 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 proteinprotein inter-

    actions, and of a C-terminal low-complexity region,

    mainly composed of polyglutamates.

    Since their rst 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 trafcking 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 [48]. 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 [912]. The N-terminal LRR


    ataxin 1; leucine-rich repeats; NMR; PP2A

    inhibitor; structure


    A. Pastore, National Institute for Medical

    Research, The Ridgeway, London NW7

    1AA, UK

    Fax: +44 208 906 4477

    Tel: +44 208 959 3666


    (Received 13 January 2008, revised 3 March

    2008, accepted 14 March 2008)


    The leucine-rich repeat acidic nuclear protein (Anp32a LANP) belongs toa family of evolutionarily-conserved phosphoproteins involved in a com-

    plex network of proteinprotein 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

    unmodied domains bind with very weak (millimolar) afnity, 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

    modication. Finally, we identied 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 microtubulesand to be associated with severe human pathologies.


    Anp32a LANP, leucine-rich repeat acidic nuclear protein; Atx1, ataxin-1; AXH, ataxin-1 homology; Gal-X, 5-bromo-4-chloroindol-3-ylb-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) 25482560 2008 The Authors Journal compilation 2008 FEBS

  • domain of Anp32 binds and strongly inhibits the

    enzyme catalytic subunit PP2A-C [37], whose struc-

    ture in a heterotrimeric complex with the scaffolding

    A subunit and the regulatory B B56 PR61 subunitwas solved recently [13,14]. Although the role of phos-

    phorylation in recognition remains debatable, interac-

    tion between Anp32a and PP2A-C has been

    independently conrmed 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. Immunouores-

    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-specic 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 rst 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 denition 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 rst solved the structure in solution of Anp32

    by NMR spectroscopy. This technique, which does not

    need crystallization, also provides a powerful and exi-

    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 denition

    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 LRRprotein 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 identied a new

    partner of the Anp32a LRR domain.


    Description of the Anp32a LRR domain structure

    in solution

    The construct used for structure determination covers

    residues 1164 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 nal representative family of the

    10 lowest-energy structures after water renement

    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 3154 (Fig. 1). The structure was

    solved at high precision and has an excellent whatif

    score (Table 1).

    The domain topology (h1h2b1b2b3h3b4h4b5h5h6b6b7b8) shows the secondary structure elements spatiallyarranged 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 theconvex surface contains both well-dened but irregular

    secondary structure elements (in the rst and second

    repeats) and helical regions. Among these, h1 and h6are regular a-helices whereas h2, h3, h4 and h5 sharefeatures of 310-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 ve com-

    plete LRRs anked by an a-helix at the N-terminusand by the C-terminal anking motif termed LRRcap

    (SMART accession number SM00446), so far identi-

    ed in several SDS22-like and typical LRR-contain-

    ing proteins, such as U2A, TAP, RabGGT, anddynein 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) 25482560 2008 The Authors Journal compilation 2008 FEBS 2549

  • residues Leu128 to Asp146, and includes h6, which

    belongs to the fth LRR, and the short strand b6,which runs parallel to b5 and is antiparallel to b7. Thesolution 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 1149

    (Fig. 2A,B). Despite the structural similarity, only four

    repeats (4465, 6689, 90114 and 115138 in our

    structure) were identied in the crystal structure [19],

    whereas the rst repeat (residues 1943 in our struc-

    ture) was considered by these authors to be an

    N-CAP. The presence of the rst 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 de full


    Residues 147149, which are truncated in the crystal

    structure, form a b-hairpin (b7) with the strand 143145 (b6). This region shares a remarkable similaritywith U2A, the two protein with the highest structuralhomology: the two proteins can be superimposed with

    2 A 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 (b8) is present in some of the NMR struc-tures in region 149164 (residues 151153). 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 proapoptoticactivity, which have been mapped to residues 150180

    and 150174, respectively [15,24,25]. Interestingly, this

    tail contains one of the two CK2 phosphorylation

    motifs (158161) 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 exible 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

    1H15N relaxation studies were carried out to assess the

    dynamic properties of the Anp32a LRR domain

    (Fig. 3). The correlation time, as estimated from the T1and T2 relaxation data using the model-free approach

    [28], is 12.5 0.1 ns at 27 C, a value within the rangeexpected for a single monomeric species of this size in

    solution [29]. The at prole of the relaxation parame-

    ters along the sequence indicates that, with the excep-

    tion of the rst two N-terminal amino acids and of the

    C-terminal tail (Ala155Val164), 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 the1H15N heteronuclear single quantum coherence

    (HSQC) spectrum at pH 7 (Met3, Asp4, Ile30, Glu31,

    Ile34, Glu35 and Val52) the last ve belong to regions

    without a regular secondary structure. All seven resi-

    dues, including Met3 and Asp4 at the N-terminus of

    h1, 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 T1 and RDC values and the secondary struc-

    ture: the concave b-sheet is characterized by shorterT1 and positive RDC values, the latter indicating that



    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) 25482560 2008 The Authors Journal compilation 2008 FEBS

  • the corresponding residues are oriented parallel to the

    external magnetic eld B0 when in the anisotropic

    medium [30]. Conversely, the NH vectors in the long

    helices (h1, h3, h4, and h6) running approximately

    parallel to each other are mainly perpendicular to the

    b-sheet vectors and, therefore, to B0 in the alignedmedium.

    These results suggest that interactions with other

    molecules involving the LRR domain are not medi-

    ated by an induced t mechanism but by semirigid

    docking of the partners onto the surface of the

    Anp32a LRR domain. Interactions with the INHAT

    complex, mapped to the exible C-terminus, may

    induce structuring and stiffening of this region.

    Modeling the interaction of Anp32a with other

    proteins on the basis of the U2AU2B structure

    The structural similarity with U2A, whose structure isknown in a complex with its target U2B [20], mayprovide 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 tting a helix

    of U2B (residues 2535) into the concave surface ofthe U2A LRR (Fig. 4). The size complementarity isalmost 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 restraintsa 5151

    Unambiguous ambiguous 3774 1376Intraresidue 2021

    Sequential 1075

    Medium (residue i to i + j, j = 14) 663

    Long-range (residue i to i + j, j > 4) 1392

    Dihedral angle restraintsb

    F 89w 891DNH RDC 92

    Hydrogen bonds 20

    Deviation from idealized geometry

    Bond lengths (A) 0.003 0.000

    Bond angles () 0.503 0.011Improper dihedrals () 1.442 0.073

    Restraint violations

    Distance restraint violation > 0.5 A 0

    Dihedral restraint violation > 5 0Coordinate precision (A) with respect to the mean structure

    Backbone of structured regionsc 0.71 0.15

    Heavy atoms of structured regionsc 1.16 0.16

    WHATIF quality checkd

    First-generation packing quality 0.01

    Second-generation packing quality )2.44Ramachandran pl...


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