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

Abstract

Spinocerebellar ataxia type 3 is a human neurodegenerative disease resulting from polyglutamine tract expansion. The affected protein, ataxin-3, which contains an N-terminal Josephin domain followed by tandem ubiquitin (Ub)-interacting motifs (UIMs) and a polyglutamine stretch, has been implicated in the function of the Ub proteasome system. NMR-based structural analysis has now revealed that the Josephin domain binds Ub and has a papain-like fold that is reminiscent of that of other deubiquitinases, despite primary sequence divergence but consistent with its deubiqutinating activity. Mutation of the catalytic Cys enhances the stability of a complex between ataxin-3 and polyubiquitinated proteins. This effect depends on the integrity of the UIM region, suggesting that the UIMs are bound to the substrate polyubiquitin during catalysis. We propose that ataxin-3 functions as a polyubiquitin chain-editing enzyme.

Fig. 1.

Tertiary structure of the JD of ataxin-3. (A) Stereoview of the superimposed Cα 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 β-sheet and two α-helices. The right lobe (red) comprises only α-helices.

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 1UCH). α-Helices are represented by filled circles, and β-strands are represented by triangles. β-Strand direction out from the paper is indicated by left-oriented triangles, and β-strand direction into the paper is indicated by right-oriented triangles. The left lobe of the JD is conserved in UCH-L3 (colored in blue). The main topological differences are observed in the right lobe. The N-terminal α-helices of the JD are “shuffled” as a big insertion between the β2 and β3 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 abbreviations. (C and D) Ribbon diagrams of the catalytic sites of the JDs of ataxin-3 (C) and UCH-L3 (D), with the active site residues shown in ball-and-stick models. The catalytic residues have a similar geometric organization in the two proteins, suggesting a similar mechanism of catalysis.

Fig. 3.

Interaction between Ub and the JD as assessed by NMR chemical shift perturbation. (A) Chemical shift perturbation of the JD upon interaction with Ub. Ribbons are colored according to whether corresponding backbone nuclei experienced primarily chemical shift changes (from gray to yellow) or loss of 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 JD of ataxin-3. The color code is the same as in A. The ribbon diagram was generated from deposited Ub structure coordinates (Protein Data Bank ID code 1UBQ).

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 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 mediated by the first two UIMs, for the longest chains. Ctr, control. (B) Deubiquitination assays carried out by incubating tetraubiquitin with recombinant His-tagged ataxin-3 and JD and their mutants. At the end of the reaction, Ub and ataxin-3 proteins were detected by Western blotting. Activity is indicated by the conversion of tetraubiquitin to triubiquitin. (C) In vivo substrate trapping by catalytically inactive ataxin-3. HeLa cells were transfected with FLAG-tagged wild-type ataxin-3 or mutant ataxin-3 constructs or with the empty vector (Ctr lane). FLAG-tagged proteins were then lysed and immunoprecipitated in JS buffer (Left) or radioimmunoprecipitation assay buffer (to disrupt Ub–UIM interactions) (Right), and the immunoprecipitates were analyzed by Western blotting for Ub or for ataxin-3 with an antibody that recognizes its JD. Arrows indicates IgG heavy chains.

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 VCP/p97, which may target ataxin-3, to function in one or more VCP/p97 pathways and/or directly mediate the regulation of the deubiquitinating activity of ataxin-3 by VCP/p97.