Insights from the Solution Structure of the Josephin Domain

Deubiquitinating function of ataxin-3: Insights from the solution structure of the Josephin domain

Yuxin Mao*, Francesca Senic-Matuglia†, Pier Paolo Di Fiore†‡§, Simona Polo†‡, Michael E. Hodsdon¶ʈ, and Pietro De Camilli*ʈ

*Howard Hughes Medical Institute and Department of Cell Biology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510; ¶Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06510; and †Istituto Fondazione Italiana per la Ricerca sul Cancro di Oncologia Molecolare, ‡Istituto Oncologico Europeo, and §Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita’ degli Studi di Milano, 20122 Milan, Italy

Contributed by Pietro De Camilli, July 25, 2005 Spinocerebellar ataxia type 3 is a human neurodegenerative components of the quality control and protein degradation disease resulting from polyglutamine tract expansion. The af- system. Ataxin-3 interacts with the valosin-containing protein fected protein, ataxin-3, which contains an N-terminal Josephin (VCP͞p97) (18, 19), a type II AAA ATPase that is associated domain followed by tandem ubiquitin (Ub)-interacting motifs with variety of cellular functions, including membrane fusion, (UIMs) and a polyglutamine stretch, has been implicated in the endoplasmic reticulum-associated degradation, and regulation function of the Ub proteasome system. NMR-based structural of the NF-␬B pathway (20). Ataxin-3 also interacts with HHR23 analysis has now revealed that the Josephin domain binds Ub (RAD23 in yeast) (19, 21), a ‘‘shuttle factor’’ responsible for and has a papain-like fold that is reminiscent of that of other translocating ubiquitinated proteins to the proteasome, and with deubiquitinases, despite primary sequence divergence but con- the proteasome itself (19). sistent with its deubiqutinating activity. Mutation of the cata- The polyQ stretch is variable in length and causes disease when lytic Cys enhances the stability of a complex between ataxin-3 expanded beyond a critical threshold. The polyQ stretch com- and polyubiquitinated proteins. This effect depends on the prises 10–40 Glu residues in unaffected individuals and 55–84 integrity of the UIM region, suggesting that the UIMs are bound Glu residues in Machado–Joseph disease patients (22). Similar to the substrate polyubiquitin during catalysis. We propose that expansions of polyQ stretches are responsible for at least nine ataxin-3 functions as a polyubiquitin chain-editing enzyme. inherited human neurodegenerative disorders, including Hun- tington’s disease. Proteins containing such expansions have an ͉ ͉ ͉ ͉ ataxia polyglutamine ubiquitin ubiquitin interaction motif increased propensity to misfold and aggregate (23–25), often valosin-containing protein forming intranuclear inclusions in affected cells. Accordingly, there is evidence that proteins involved in the control of protein pinocerebellar ataxia type 3, also known as Machado–Joseph folding and degradation can affect disease progression in animal Sdisease, is one of several hereditary autosomal dominant models (26, 27). The functional link of ataxin-3 to the ubiquitin- neurodegenerative disorders caused by expansion of a polyglu- proteasome system and to VCP͞p97 suggests a potential inter- tamine (polyQ) stretch in the affected gene product (1, 2). The play between the properties of these proteins and the pathogenic gene responsible for spinocerebellar ataxia type 3 encodes a role of its polyQ expansion. In fact, a very recent study demon- 42-kDa protein named ataxin-3. Ataxin-3 contains an N- strated that functions of ataxin-3 directly related to its property terminal Josephin domain (JD), a conserved module named to bind and process polyubiquitin suppress polyQ-induced de- after the Machado–Joseph disease, two ubiquitin (Ub)- generation in Drosophila (28). interacting motifs (UIMs), a polyQ stretch, and a short variable The proposed role of ataxin-3 as a deubiquitinating enzyme tail. In one splice variant, the C-terminal region ends with a third raises the question of a potential coordination between such putative UIM (3). catalytic activity and polyubiquitin binding by the UIMs. For The JD is present in at least 30 predicted proteins, including example, UIMs may help to recruit ataxin-3 to polyubiquitinated two human proteins comprising the JD alone. Computational substrates, orient such substrates relative to the JD, or mediate analyses resulted in two different predictions about the JD function. One study noted a distant structural homology to the a polyubiquitin editing function of ataxin-3. In addition, given epsin N-terminal homology͞AP180 N-terminal homology do- the lack of primary sequence similarity of ataxin-3 to other mains (4–6) present in adaptor proteins implicated in endocy- deubiquitinating enzymes, it would be of interest to elucidate the tosis (7). Another study identified signature motifs for Cys structural basis of its enzymatic activity. To begin addressing proteases in the JD and suggested that it may function as a these questions, we have performed NMR studies and biochem- deubiquitinating enzyme (8). Indeed, ataxin-3 has been shown ical analyses. We report that the tertiary structure of the ataxin-3 recently to have deubiquitination activity, which was abolished JD is strikingly similar to other deubiquitinating enzymes, such by mutation of a Cys (C14) in the putative catalytic site (9, 10). as UCH-L3. Our results also suggest cooperation between the A recent classification defined the ataxin-3͞Josephin family as JD and the UIMs during catalysis and are consistent with an one of five distinct families of deubiquitinating enzymes (11). editing function for this enzyme. Collectively, our results define The UIM is a conserved 15-aa motif first discovered as a polyubiquitin binding domain in the S5a proteasome subunit (12). This motif was subsequently identified in proteins involved Freely available online through the PNAS open access option. in endocytic traffic, as well as in other components of the Ub Abbreviations: JD, Josephin domain; NOE, nuclear Overhauser effect; polyQ, polyglutamine; Ub, ubiquitin; UIM, Ub-interacting motif; VCP, valosin-containing protein. proteasome system, such as deubiquitinating enzymes and Ub Data deposition: The JD chemical shift assignments have been deposited in the BioMagRes- ligases (13–15). Through its UIMs, ataxin-3 binds to polyubiq- Bank (BMRB accession no. 6742). Coordinates for the ensemble of the JD structures have uitin chains but not to diubiquitin or monoubiquitin (16, 17), been deposited in the Protein Data Bank (PDB ID code 2AGA). suggesting that it may function in the protein surveillance ʈTo whom correspondence may be addressed. E-mail: [email protected] or pathway leading to the proteasome. This possibility is further [email protected]. supported by the reported interaction of ataxin-3 with other © 2005 by The National Academy of Sciences of the USA

12700–12705 ͉ PNAS ͉ September 6, 2005 ͉ vol. 102 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506344102 Downloaded by guest on September 26, 2021 ataxin-3 as a critical component of the polyubiquitin-dependent Restraints on the backbone dihedral angles were derived from pathways that control protein folding and stability. an analysis of backbone chemical shifts with the TALOS program (31). Hydrogen bonds were identified during the later stages of Materials and Methods structure determination based on the consistent proximity of Antibodies and Reagents. Antibodies against ataxin-3 were gen- hydrogen bonding partners in the calculated ensembles. Nuclear erated in rabbits by using recombinant GST-JD (amino acids Overhauser effect (NOE) correlations between nearby protons 1–198) and affinity-purified. Monoclonal anti-Ub antibodies were identified in 3D 15N-NOESYHSQC, 13C-NOESYHSQC (P4D1) were from Santa Cruz Biotechnology, and monoclonal (aromatic), and 13C-NOESYHSQC (aliphatic) NMR spectra. anti-Flag M2 antibodies were from Sigma. Structure calculations were ultimately performed with the CYANA software package (32) based on NOEs that were inter- Cloning, Mutagenesis, and Protein Preparation. A cDNA encoding preted and calibrated by the program CANDID (33). Manually full-length human ataxin-3 (Machado–Joseph disease 1-1 splice interpreted NOE restraints based primarily on the use of sym- variant carrying a polyQ sequence of 22 residues) was amplified metry-related 3D NOE crosspeaks during the iterative cycles of by PCR with Integrated Molecular Analysis of Genomes and NOE interpretation and structure calculation were added to Their Expression (I.M.A.G.E.) clone ID 4393766 as a template. assist structural convergence. The final series of structure cal- The cDNA was subcloned in pGEX-6P-1 and pET43 for expres- culations used the CANDID-derived NOE restraints and the sion in bacteria and in pcDNA-FLAG vectors for mammalian previously described hydrogen bond and backbone torsion angle expression. The JD domain from the same proteins (amino acids restraints. After an initial brief minimization of randomized 1–193) was obtained in a similar way. All of the mutants were conformations, simulated annealing began with 5,000 steps of generated by site-directed mutagenesis, and the constructs were molecular dynamics at high temperature, followed by 35,000 verified by sequencing. dynamics steps for cooling and a final 10,000 steps of conjugate GST and His-6 fusion proteins were produced according to gradient minimization. Generally, 50 structures were indepen- manufacturer’s instructions. For NMR sample preparation, the dently calculated, and the 20 structures with the lowest target JD protein was expressed as a GST fusion. Isotopically labeled function values were retained as the final ensemble. protein was produced by using the appropriate Spectra-9 bac- terial growth medium. The GST tag was removed by PreScission Ub Binding by NMR Chemical Shift Perturbation. 15N- and 13C15N- protease from the fusion proteins bound to glutathione beads, labeled JD at 100 ␮M was titrated with unlabeled Ub in and the eluted protein was further purified with size exclusion concentrations from 33 to 2,400 ␮M. Two-dimensional 1H15N chromatography. NMR samples of 1.5 mM JD were prepared in HSQC spectra were measured for each condition and 3D HNCO a 20 mM potassium phosphate, pH 6.4͞0.05% azide͞5% NMR spectra were collected for samples at 1:0 and 1:2 molar 2 1 15 H2O͞10 ␮M 2,2-dimethyl-2-silapentane-5-sulfonate solution ratios of Josephin and Ub, respectively. Vice versa, 2D H N containing 1 ␮M of the protease inhibitors PMSF, leupeptin, and HSQC spectra from [15N]Ub alone or [15N]Ub mixed with pepstatin. unlabeled JD were also collected. NMR resonances for the JD were affected in one of three ways: (i) none or minimal change Pull-Downs and Deubiquitination Assays. For pull-down experi- in intensity or chemical shift, (ii) major perturbation of chemical ments, His-tagged ataxin-3 proteins were incubated for2hat4°C shift with minor intensity reduction, or (iii) major intensity with 400 ng of K48-linked polyubiquitin chains and Ni2ϩ beads reduction or ‘‘missing’’ crosspeaks due to extreme line broad- in 50 mM Hepes, pH 7.4͞10% glycerol͞1% Triton X-100͞1mM ening. Because of the transient nature of the interaction between EDTA͞1 mM EGTA͞10 mM MgCl2͞10 mM CaCl2͞20 mM Ub and the JD, association–dissociation exchange of Ub is likely imidazole. Bound material was analyzed by Western blotting. to occur on a time scale similar to the NMR chemical shift For deubiquitination assays, 300 ng of tetraubiquitin was differences between the two states. For the largest chemical shift incubated with 100 pmol of His-tagged ataxin-3 proteins at 37°C differences, NMR line shapes are broadened because of the in 50 mM Tris, pH 7.4͞5 mM MgCl2͞25 mM KCl͞1mMDTT. contribution of the exchange process to NMR relaxation, with After 18–22 h (the reaction occurred very slowly), the reaction the largest chemical shift perturbations resulting in the broadest was stopped with SDS sample buffer and samples were analyzed peaks. Hence, for the purpose of constructing Fig. 3, chemical by Western blotting. shift perturbations were scaled from gray to yellow for class II, based on the weighted sum of the chemical shift changes for the Transfection and in Vivo Binding Experiments. HeLa cells were amide 1H, 15N, and carbonyl 13C nuclei, and then from yellow to transfected with ataxin-3 constructs by using Lipofectamine orange for class III, based on the relative decrease in peak according to the manufacturer’s protocol. Cells were harvested heights due to line broadening. Three well-resolved peaks in the 24 h later and lysed in JS buffer (50 mM Hepes͞150 mM 2D 1H15N HSQC spectra experiencing significant chemical shift NaCl͞10% glycerol͞1% Triton X-100͞1.5 mM MgCl2͞5mM changes during the titration were selected for simultaneously EGTA, pH 7.5) or in radioimmunoprecipitation assay buffer (50 fitting to a single-site model of ligand-binding using SIGMAPLOT mM Tris͞150 mM NaCl͞1% Triton X-100͞1% deoxycolate͞ to estimate a dissociation constant of 3.4 Ϯ 0.3 mM. 0.1% SDS, pH 7.4). Cleared lysate (1 mg) was then incubated with anti-FLAG M2 gel for 2 h. After extensive washing, bound Results proteins were analyzed by Western blotting. NMR Solution Structure of the JD. The solution structure of the JD (amino acids 1–193 of human ataxin-3) (Fig. 6, which is pub-

NMR Resonance Assignments and Structure Determination. All NMR lished as supporting information on the PNAS web site) was BIOCHEMISTRY spectra were collected at 25°C on a Varian INOVA 600 MHz determined by NMR spectroscopy. As a first step toward struc- spectrometer and used pulse sequences available in the Varian ture determination, the 1H, 13C, and 15N NMR chemical shifts of BioPack User Library. Sequential and aliphatic side-chain as- the JD were assigned to near completion. Chemical shifts for the signments were determined by analysis of 3D-HNCO, HNCA, last 10 aa were observed in freshly prepared samples, but the HNCACB, HN(CO)CA, HCACO, CBCA(CO)NH, (HCA)CO- chemical shifts diminished over time, probably because of pro- (CA)NH, CC(CO)NH, HCC(CO)NH, and HCCH-total corre- teolysis as the emergence of new NMR signals characteristic of lation spectroscopy NMR experiments. All spectra were pro- degraded peptides. Thus, the last 10 aa were not included in the cessed with NMRPIPE (29) and subjected to visual analysis in final structure calculations. A secondary set of chemical shifts SPARKY (30). was observed for a contiguous stretch of 22 residues (G51–G72).

Mao et al. PNAS ͉ September 6, 2005 ͉ vol. 102 ͉ no. 36 ͉ 12701 Downloaded by guest on September 26, 2021 Fig. 1. Tertiary structure of the JD of ataxin-3. (A) Stereoview of the superimposed C␣ backbone traces of the 20 lowest-energy calculated structures. Traces are rainbow-colored, with the N terminus (N) in red and the C terminus (C) in blue. Numbers and corresponding small ﬁlled circles indicate residue multiples of 20. (B) Ribbon diagram of a representative structure from A shown in the same orientation. The left lobe (blue) comprises a six-stranded ␤-sheet and two ␣-helices. The right lobe (red) comprises only ␣-helices.

The peak intensities of these secondary shifts were significantly comprises three of the four families of deubiquitinating enzymes smaller than those corresponding to the predominant confor- whose structures have already been characterized. Among pro- mation and had many fewer NOE crosspeaks. Thus, the 22 amino teins with the highest Z scores (Ϸ5.0) is the Ub C-terminal acids were omitted from the final structure calculations. The existence of the secondary set of chemical shifts likely reflects an alternative conformation for these residues, which may be important for protein function (discussed further below). Inde- pendent chemical-shift assignments for the ataxin-3 JD were recently published (34). Although chemical shifts for all of the backbone atoms and for the majority of the side chains are identical to ours, there are differences for side-chain atoms in several residues, which are most likely caused by differences in experimental conditions, such as pH. Tertiary structures were calculated with the CYANA software package (32, 33) based on a total of 2,960 NOE-derived distance restraints, 122 hydrogen bond restraints, and 224 backbone torsion angle restraints. The final ensemble of tertiary structures is presented as a stereo pair in Fig. 1A and structural statistics are listed in Table 1, which is published as supporting information on the PNAS web site. The JD adopts an ␣͞␤ fold consisting of a six-stranded, antiparallel ␤-sheet (␤1–␤6) flanked on both sides by a total of seven ␣-helices (␣1–␣7) (Fig. 1). The overall structure has a bi-lobed appearance divided by a groove. One lobe (in red and at the right in Fig. 1B) contains five ␣-helices (␣1–␣4 and ␣7). ␣1 and ␣4 are almost parallel to each other and packed by means of strong hydrophobic interactions against the ␤-strands. The much longer ␣2 is oriented nearly perpendicular to ␣1 and ␣4 at the surface of the domain. The stretch of residues comprising the short ␣3 and the following loop are associated with two alternative sets of chemical shifts and are therefore likely to undergo dynamic conformational changes. The other Fig. 2. Structural comparison of the JD with UCH-L3. (A and B) Secondary ␣ structure topologies of the JD (A) and UCH-L3 (B) (Protein Data Bank ID code lobe (in blue and at the left in Fig. 1B) consists of two helices ␣ ␤ ␣ ␣ ␤ 1UCH). -Helices are represented by ﬁlled circles, and -strands are repre- ( 5 and 6) and the six-stranded sheet. The six antiparallel sented by triangles. ␤-Strand direction out from the paper is indicated by ␤-strands have an average length of four residues with the left-oriented triangles, and ␤-strand direction into the paper is indicated by following sequence: ␤1, ␤6, ␤2, ␤3, ␤4, and ␤5. The gaps between right-oriented triangles. The left lobe of the JD is conserved in UCH-L3 (colored ␤1 and ␤2 and ␤5 and ␤6 are filled by ␣5 and ␣6, respectively, in blue). The main topological differences are observed in the right lobe. The whereas the connections between ␤2, ␤3, ␤4, and ␤5 are made N-terminal ␣-helices of the JD are ‘‘shufﬂed’’ as a big insertion between the ␤2 by three tight turns. and ␤3 strands in UCH-L3. The ‘‘substrate-limiting loop,’’ which crosses over the catalytic site in UCH-L3 (yellow), is missing in the JD. Green letters indicate the positions of the catalytic residues with single-letter amino acid abbrevia- The JD Resembles Deubiquitinating Enzymes But Has Unique Structural tions. (C and D) Ribbon diagrams of the catalytic sites of the JDs of ataxin-3 (C) Features. A search for homologous structures using the DALI and UCH-L3 (D), with the active site residues shown in ball-and-stick models. algorithm (35) showed that the JD is structurally similar to The catalytic residues have a similar geometric organization in the two pro- proteins in the papain-like Cys–protease family. This family teins, suggesting a similar mechanism of catalysis.

12702 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506344102 Mao et al. Downloaded by guest on September 26, 2021 may be the result of direct interactions with Ub or indirect structural changes due to complexation. Most of the perturbed residues cluster into a single contiguous group comprising ␣3, the loop between ␣3 and ␣4, and the C-terminal portion of ␣2. These results suggest a general location for the Ub-binding site above the catalytic site as oriented in Fig. 3A, which is in agreement with the previously reported complex of Yuh1 and herpesvirus-associated Ub-specific protease with Ub aldehyde (38, 39). The existence of an alternative conformation or dynamic state for ␣3 and the ␣3–␣4 loop combined with their dramatic chemical shift changes may reflect an inducible structural ordering for these residues associated with Ub binding. We also collected 2D 1H15N HSQC spectra from [15N]Ub alone or [15N]Ub mixed with unlabeled JD. Residues 5–15, 29–36, and 68–75 of Ub displayed the largest chemical-shift perturbations. Fig. 3. Interaction between Ub and the JD as assessed by NMR chemical shift When mapped onto the Ub surface, these chemical-shift- perturbation. (A) Chemical shift perturbation of the JD upon interaction with perturbed residues form a contiguous region (Fig. 3B). A similar Ub. Ribbons are colored according to whether corresponding backbone nuclei surface area of Ub has also been reported by NMR chemical- experienced primarily chemical shift changes (from gray to yellow) or loss of shift perturbation to be involved in binding to UCH-L3 (40). signal intensity (from yellow to orange) as detailed in Materials and Methods. The position of the catalytic site is marked by dashed-line circle. C, C terminus; N, N terminus. (B) Chemical shift perturbation of Ub upon interaction with the The UIMs of Ataxin-3 Are Involved in the Deubiquitinating Reaction. JD of ataxin-3. The color code is the same as in A. The ribbon diagram was Ataxin-3 has at least two other Ub-binding sites that are the generated from deposited Ub structure coordinates (Protein Data Bank ID tandem UIMs immediately C-terminal to the JD. A plausible code 1UBQ). speculation is that these UIMs recognize the polyubiquitinated substrate and help position the polyubiquitin chain close to the catalytic site on the JD, whereas the Ub-binding site on the JD hydrolase UCH-L3, which has the lowest rms deviation value of ␣ transiently holds the distal Ub during catalysis (see Fig. 5). To 3.2 Å for all C atoms. Comparison of the structural topologies test this idea, we performed in vitro and in vivo binding assays. of the JD and UCH-L3 reveals that the lobe containing the Pull-down assays of polyubiquitin mixtures on ataxin-3 con- ␤ -sheet (at left and colored in blue in Figs. 1 and 2) is structurally structs demonstrated that, in agreement with previous data (16, ␣ conserved. In contrast, the other lobe, comprising only -helices, 17), full-length ataxin-3 interacts with K48-linked polyubiquitin has a different organization of secondary structure elements chains with a preference for high-molecular-weight chains (n Ն relative to UCH-L3. Another major distinction involves the 3). As shown by mutation of the Ser critically required for connection between ␤2 and ␤3, which, in the JD, is represented binding in the UIMs, this interaction largely depends on the first by a tight ␤-turn, whereas, in UCH-L3, it comprises a large and second UIMs (17), although it is independent from the third insertion including ␣3, ␣4, ␣5, and ␣6. putative UIM (Fig. 4A). Despite this clear interaction in cell-free Our results demonstrate the validity of a previous bioinfor- assays, no significant coprecipitation between ataxin-3 and matics analysis that had identified some weak similarities be- polyubiquitinated substrates could be obtained from cell lysates. tween the regions surrounding the catalytic residues His, Asn͞ The binding of ataxin-3 to its substrates may therefore be very Asp, and Cys of deubiquitinating enzymes and three short transient in vivo. However, if the UIMs of ataxin-3 interact with segments in ataxin-3 (Fig. 6) (8, 36). In the JD structure, the the polyubiquitin chain during catalysis, mutations that affect the predicted catalytic residues (C14, H119, and N134) are indeed in catalytic activity of the JD but not its Ub-binding sites are close proximity to each other, forming the so-called catalytic expected to enhance the stability of the interaction between triad, which is characteristic of Cys proteases (Fig. 2B). The ataxin-3 and its substrate (see Fig. 5). Under these conditions, organization of the catalytic triad residues is in an appropriate the polyubiquitin chain would be held attached to ataxin-3 by geometry for catalytic function. Thus, these structural data are two anchorage points (the UIMs and the Ub-binding site of the consistent with the reported deubiquitinating activity of ataxin-3 JD) at both sides of the inactive catalytic site. (9, 37). We tested this possibility. First, with tetraubiquitin as a Based on the conserved topology of the catalytic site residues, substrate, we confirmed that, although purified full-length a likely mode of Ub binding to the JD can be proposed by ataxin-3 and its isolated JD have deubiquitinating activity, analogy with the structures of the deubiquitinating enzyme Yuh1 their C14A mutants do not (9, 37) (Fig. 4B). Next, we analyzed (the yeast orthologue of UCH-L3) and herpesvirus-associated immunoprecipitates from cells transfected with FLAG-tagged Ub-specific protease in a complex with the inhibitor Ub aldehyde wild-type and mutant ataxin-3 constructs for the presence of (38, 39). The distal Ub may interact with the area above the coprecipitating ubiquitinated proteins by Western blotting. catalytic Cys and His residues (in the orientation of the protein Immunoprecipitates generated from cells expressing ataxin- shown in the diagrams of Figs. 2 and 3). The extended C terminus 3C14A contained much higher levels of ubiquitinated proteins of the Ub would then fit in the groove between the six-stranded relative to those generated from cells expressing wild-type sheet on the left and the variable ␣-helices on the right. This ataxin-3 (Fig. 4C, lane 2 and 3), and coprecipitation was

mode of binding would place the C-terminal G76 of Ub next to abolished if cells were lysed in radioimmunoprecipitation assay BIOCHEMISTRY the catalytic Cys (C14) of the JD. We tested this prediction by buffer, i.e., under conditions that disrupt UIM binding to Ub performing NMR chemical shift perturbation experiments to (Fig. 4C, lane 10 and 11). These data strongly suggest the map the structural interactions between the JD and Ub. trapping of an enzyme–substrate complex. Furthermore, this effect required portions of ataxin-3 outside the JD, because a Interaction of the JD with Ub. Two-dimensional 1H15N HSQC and corresponding mutant JosephinC14A domain did not retain 3D HNCO NMR spectra from the 13C15N-JD alone or mixed ubiquitinated proteins (Fig. 4C, lanes 4 and 5). Furthermore, with unlabeled Ub were collected and compared. Changes in we found that the first and third UIMs are almost dispensable backbone NMR chemical shifts were then mapped onto the for substrate trapping, because ataxin-3C14A, S236A and ataxin- surface of the JD (Fig. 3A). These chemical shift perturbations 3C14A, S347A still efficiently pulled down ubiquitinated proteins

Mao et al. PNAS ͉ September 6, 2005 ͉ vol. 102 ͉ no. 36 ͉ 12703 Downloaded by guest on September 26, 2021 Fig. 5. Putative model of ataxin-3 function. Several functional groups are arranged in a linear fashion in ataxin-3. The Ub-binding site in the JD is followed in order by the catalytic site, the tandem UIMs, the polyQ stretch, and the variable C terminus that may contain a putative UIM. The tandem UIMs may recruit a polyubiquitinated substrate and present it to the JD. The JD holds a distal Ub in a tetrahedral intermediate state before releasing it after catalytic cleavage. The model implies that optimal substrates of ataxin-3 should have a minimum polyubiquitin chain length to allow engagement of both the second UIMs and the JD in Ub binding. Thus, the model is consistent with a role of ataxin-3 as a polyubiquitin-editing enzyme. The dotted lines connecting Ubs indicate the potential variable length of the polyubiquitin chain. The sequence around the polyQ stretch is responsible for the binding of ataxin-3 to VCP͞p97, which may target ataxin-3, to function in one or more VCP͞p97 pathways and͞or directly mediate the regulation of the deubiquitinating activity of ataxin-3 by VCP͞p97. Fig. 4. Functional interplay between the catalytic activity of the JD of ataxin-3 and its UIMs during catalysis. (A) Pull-down of synthetic K48-linked polyubiquitin chains onto recombinant His-tagged wild-type and mutant tured, with no significant interactions between this region and ataxin-3 (Atx) and isolated JD. Bound polyubiquitin and the bait proteins were detected by Western blotting (WB). Note the preference of ataxin-3, primarily the N-terminal JD (25). Together with these previous results, mediated by the ﬁrst two UIMs, for the longest chains. Ctr, control. (B) our findings support a model in which the functional groups of Deubiquitination assays carried out by incubating tetraubiquitin with recom- ataxin-3 are arranged in an extended linear fashion, starting binant His-tagged ataxin-3 and JD and their mutants. At the end of the with the Ub-binding site of the JD and followed in order by the reaction, Ub and ataxin-3 proteins were detected by Western blotting. Activity catalytic triad, the tandem UIMs, the polyQ stretch, and the is indicated by the conversion of tetraubiquitin to triubiquitin. (C) In vivo variable C terminus. This arrangement could accommodate a substrate trapping by catalytically inactive ataxin-3. HeLa cells were trans- polyubiquitin chain with a (distal) Ub bound to the Ub- fected with FLAG-tagged wild-type ataxin-3 or mutant ataxin-3 constructs or interacting site on the JD, its C-terminal isopeptide bond with the empty vector (Ctr lane). FLAG-tagged proteins were then lysed and immunoprecipitated in JS buffer (Left) or radioimmunoprecipitation assay juxtaposed to the catalytic site, and proximal Ub(s) bound to buffer (to disrupt Ub–UIM interactions) (Right), and the immunoprecipitates the UIMs (Fig. 5). In this fashion, these multiple functional were analyzed by Western blotting for Ub or for ataxin-3 with an antibody units act in a concerted way in the deubquitinating reaction. that recognizes its JD. Arrows indicates IgG heavy chains. This model allows the proper orientation of the catalytic substrate on the surface of ataxin-3 for the formation of an enzyme–Ub tetrahedral intermediate, as observed in the (Fig. 4C, lanes 6 and 9). However, mutations in the second structures of UCH-L3 and herpesvirus-associated Ub-specific S256A S236, S256A ataxin-3 or the first and second UIMs of ataxin-3 protease complexed with Ub aldehyde (38, 39). The model is drastically inhibited substrate trapping (Fig. 4C, lanes 7 and 8), also consistent with our in vivo substrate trapping data showing suggesting a critical role of the second UIM in this phenom- that the UIM region plays an important role in the catalytic enon. These ‘‘substrate trapping’’ experiments strongly indi- activity of ataxin-3. The UIMs may help to recruit polyubiq- cate a tight link between catalytic activity of the JD and uitinated substrates, to position the polyubiquitin chain rela- polyubiquitin chain binding by the UIM(s). tive to the catalytic site, and͞or to allow the enzyme to act in trimming polyubiquitin chains in a distal to proximal direction. Discussion Accordingly, preliminary results indicate that full-length Based on computational modeling, ataxin-3 was predicted to be ataxin-3 prefers to cut the distal Ub of a short Ub chain. a distant homolog of endocytic adaptins, such as epsin and An interaction between ataxin-3 and VCP͞p97 was first AP180 (7), or of deubiquitinating enzymes (8). Subsequently, suggested by the observation that VCP colocalized with ex- this domain was shown to have deubiquitinating activity (9). panded polyQ aggregates in cultured cells (18). Subsequently, a With the determination of the solution structure of the JD, direct interaction between VCP͞p97 and ataxin-3, which is ataxin-3 can be conclusively defined as a deubiquitinating mediated by the C-terminal region of ataxin-3, including its enzyme. polyQs stretch, was reported (18, 41). Indeed, not only have we Ataxin-3 is unusual among the many deubiquitinating en- confirmed that the two proteins interact in pull-down experi- zymes because of the unique array of functional units related ments, but we have also found that ataxin-3, but not its isolated to protein quality control and degradation that are contained JD, coprecipitates with VCP͞p97 from cotransfected cells (our in its sequence. It is of interest to establish how these various unpublished observations). VCP͞p97 mediates multiple cellular units cooperate. Previous data have shown that the entire functions, including membrane fusion, endoplasmic reticulum- portion of ataxin-3 distal to the JD is flexible and nonstruc- associated degradation, and other protein quality control activ-

12704 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506344102 Mao et al. Downloaded by guest on September 26, 2021 ities (20). Furthermore, recent data suggest that CDC48 (the a large rotation in the orientation of ␣3 relative to ␣2. Both studies found yeast orthologue of VCP͞p97) plays a central role in guiding evidence for structural mobility of ␣3, which is consistent with the proteolytic substrates to the proteasome for degradation (42). observation of variable conformations. These findings speak to a role of the deubiquitinating activity of We thank Drs. Randall N. Pittman (University of Pennsylvania School ataxin-3 in a pathway leading to proteasomal degradation, which ͞ of Medicine, Philadelphia), Henry L. Paulson (University of Iowa, Iowa is centered at VCP p97. City), Phyllis I. Hanson (Washington University School of Medicine, St. In conclusion, our findings demonstrate that ataxin-3 is indeed Louis), and Graham Warren (Yale University School of Medicine) for a deubiquitinating enzyme and suggest that it functions as a reagents and Drs. Arthur Horwich, Mark Hochstrasser, and Hong Chen polyubiquitin chain-editing enzyme that shortens Ub chains. for discussion and advice. Y.M. is a Howard Hughes Medical Institute These properties distinguish ataxin-3 from other deubiquitinat- Fellow of the Life Sciences Research Foundation. F.S.-M. is supported by an Associazione Italiana per la Ricerca sul Cancro Fellowship. This ing enzymes that function in the processing of Ub precursors, the work was supported in part by National Institutes of Health Grants reversal of Ub conjugation, or the recycling of Ub. NS36251 (to P.D.C.) and CA108992 (to M.E.H.) and by grants from the Human Frontiers Science Program (to P.D.C. and P.P.D.F.); the Asso- Note. Shortly before submission, an independent solution structure of ciazione Italiana per la Ricerca sul Cancro (to P.P.D.F. and S.P.); the the ataxin-3 JD was published in PNAS (43). Comparison to the European Community; the Italian Ministry of Health; the Italian structure presented here reveals striking similarities in overall global Ministry of Education, University, and Research; the Monzino Foun- conformation, with one significant and interesting difference involving dation (to P.P.D.F.); and the Charlotte Geyer Foundation (to M.E.H.).

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