AT3-UIM/Ub Interaction Figure 6. The Cooperative Interaction of AT3-UIM12 with K48- and K63- Linked diUb Chains. A, Pull-down of K48- and K63-linked diUb by GST-fused UIM1, UIM2 or UIM12. The pull-down samples were detected by Western blotting with an anti-Ub antibody. B, SDS-PAGE of the GST- fused proteins (AT3-UIM1, AT3-UIM2 and AT3-UIM12) detected by Coomassie staining. doi:10.1371/journal.pone.0013202.g006 the flexible linker (Fig. 2A & 2B), they become relatively compact and orientated when the UIM motifs bind with Ub (Figure 2C & 2D). Meanwhile, the linker region is more ordered in the complex form (Fig. 3). These two oriented helices in the Ub-bound form are likely beneficial to the binding with diUb, polyUb or ubiquitinated substrates, no matter whether K48- or K63-linked (Fig. 8C). This cooperation mode is also observed in the tandem WW domains of HYPA/FBP11 binding with N- and C-portions of proline-rich region in huntingtin (Htt) [41], and the tandem PHD and bromodomains in sumoylation and gene regulation [49]. Sterical- ly, in this binding mode, the configuration of two Ub units in diUb is presumed to match the orientation of the helical orientation of AT3-UIM12. Thus, we propose that the cooperation is not only crucial but also beneficial to the binding of AT3 protein with polyUb or multiUb, and to the function as a deubiquitinating enzyme. The in vitro binding data demonstrated striking differences between UIMs in their protein-protein interactions despite their small size and highly conserved core residues. Recent work investigated the inhibiting activity of UIM to the aggregation of Htt, and demonstrated that different UIM has different effect on the polyQ-expanded aggregates [50]. The UIMs of AT3, S5a, Eps15 and Hrs could be recruited to the aggregate, whereas the UIMs of epsin, HSJ1 and USP25 did not co-localize with Htt- 65Q-CFP. Only the expression of AT3 and S5a UIMs can decrease aggregation of Htt-65Q, but they have distinct effects on the aggregate formation. In the case of AT3, the third UIM, along with UIM1 and UIM2, works in combination for reducing aggregate formation [50]. Since the isolated single UIMs might serve as potential inhibitors of polyQ aggregation in vivo, the detailed investigation into UIM-Ub interaction will be helpful to understand the mechanism. In summary, we have solved the solution structures of AT3- UIM12 in both free- and Ub-bound forms and elucidated its binding properties with various Ub chains including monoUb, diUb and polyUb. Both AT3-UIM12 and the single UIM motifs can interact with the hydrophobic patch of Ub through its conserved hydrophobic residues. The tandem motifs show higher binding affinity for mono- or polyUb than any individual UIMs. Also, AT3-UIM12 may experience a structural transformation and dynamics change during Ub binding. The linker region does not participate in the direct binding, but the chain rigidity significantly increases when it binds to Ub. Taken together, we propose that the tandem UIM motifs in AT3 interacting with Ub chains undergo a cooperative manner that may modulate its binding efficiency with monoUb, polyUb or ubiquitinated substrates, which may probably be beneficial to selectively regulating the function of AT3 protein in cell. Materials and Methods Construction of the Plasmids The plasmid pPROEX HTa-ataxin-3 was kindly provided by Peter Breuer at Max-Planck-Institute for Biochemistry. All the subclones of tandem AT3-UIM12 (residues 222–263) and its linker mutants (GS-Sub, GS-Ins, RAP80-Linker), and single UIM motifs, AT3-UIM1 (residues 222–241), AT3-UIM2 (residues 242–261) and AT3-UIM3 (residues 335–354) were constructed via PCR method with the above AT3 as template. The PCR products were ligated into the pGBTNH or pGEX-4T vector with BamH I and Xho I restriction sites to produce GB1-fused or GST-fused UIM proteins [51]. The mutants of AT3-UIM12 (I240A, I240P and Q238E/E239T) were prepared using PCR site-directed mutagen- esis technique. The cDNA encoding Ub was cloned into pET-3a plasmid using the Nde I/BamH I sites. All the plasmids were verified by DNA sequencing. Protein Expression and Purification The tandem AT3-UIM12 was overexpressed in E. coli BL21 (DE3) strain (Invitrogen). Bacterial cells were grown at 37uC in LB media and the expression was induced by adding IPTG to a final concentration of 0.2 mM. The culture was incubated for an additional 10 h or overnight at 22uC. All GB1-fused UIM proteins were initially purified by a Ni2+-NTA column (Qiagen), followed by size-exclusion chromatography (Superdex-75, GE Healthcare). 15N- and 15N/13C-labeled UIM proteins were overexpressed in M9 PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13202