Figure 3. Backbone Dynamics of AT3-UIM12 in the Free and Ub-Bound Forms. A, The 15N-{1H}-NOE values of backbone amides are plotted against the residue number of AT3-UIM12 in the absence Ub. B, The 15N-{1H}-NOE values in the presence of two-fold amount of Ub. The secondary structures of AT3-UIM12 are indicated as narrow bars. doi:10.1371/journal.pone.0013202.g003 hetero-nuclear NOE values of 0.2,0.4, whereas the linker residues (Glu239, Ile240 and Asp241) give negative NOEs (Fig. 3A). The negative NOEs indicate that this region is much more flexible than other regions. Remarkably, these linker residues in the complex display positive NOE values (,0.2) (Fig. 3B), suggesting that the internal motions of the linker region get restricted when bound with Ub. At the same time, the two helical regions also exhibit intensity enhancements, thus it is probably that the entire molecule becomes more rigid. Based on these observations, we propose that the two UIM motifs can move freely with diverse orientations in the free form, but upon Ub binding, the linker as well as the two UIM helices become rigid in the complex [31]. Formation of the relatively orientated UIM1 and UIM2 upon Ub binding thus improve the affinity of AT3- UIM12 with Ub efficiently. Interactions of Tandem UIM12 and Individual UIMs with Ub We also investigated the interaction between the tandem UIM motifs and Ub by GST pull-down assay. The result shows that AT3-UIM12 can bind with GST-Ub, while the third UIM motif (AT3-UIM3) cannot (Supplemental Fig. S3). To map the binding interfaces between AT3-UIM12 and Ub, we collected a series of 1H-15N HSQC spectra on the 15N-labeled AT3-UIM12 in the presence of increasing amount of Ub (Supplemental Fig. S4A). A AT3-UIM/Ub Interaction reciprocal experiment was also carried out on 15N-labeled Ub titrated with AT3-UIM12. The profile for the amide chemical shift changes (Dd) of 15N AT3-UIM12 upon titration of Ub at a molar ratio of 1:2 is shown in Fig. 4A. Surprisingly, besides the conserved residues in the two helices, such as Leu229 and Leu249, Ala232 and Ala252, and Ser236 and Ser256, the chemical shifts of the linker residues (Gln238, Glu239 and Ile240) are also strongly perturbed. The large chemical shift changes imply that the linker region experiences structural and dynamics change or residue contacts upon Ub binding. The Dd values upon titration of Ub were extracted from the HSQC spectra of at different molar ratios of AT3-UIM12 to Ub. The chemical shift changes for the equivalent residues, Leu229 in UIM1 and Leu249 in UIM2, respectively, exhibit different saturation curves as Ub titration proceeds (Fig. 4B). Similar results are also observed in Ser236 and Ser256, as well as in Ala232 and Ala252 (Supplemental Fig. S4B). The Dd values of the residues in UIM2 upon Ub binding reach the plateau with lower amount of Ub than the corresponding ones in UIM1, suggesting that the two UIM motifs bind Ub with different affinities. It is likely that, in AT3-UIM12, the UIM2 moiety may have a higher Ub-binding affinity than UIM1 (Fig. 4B and Supplemental Fig. S4B). Similarly, titration of 15N-labeled Ub with AT3-UIM12 causes significant but selective perturbations on the Ub spectra (Fig. 4C). Ub specifically binds with AT3-UIM12 through its typical hydrophobic patch mainly comprised of Leu8, Ile44 and Val70. We have determined the apparent dissociation constant (KD,app) from the three strongly perturbed residues, Ile13, Lys48 and Leu71 (Fig. 4D). The apparent KD value is 97.0616.9 mM for AT3-UIM12 binding with Ub (Table 1). In AT3-UIM12, both UIM1 and UIM2 moieties can bind to Ub individually but with different affinities. It is reasonable to raise such a question as why AT3 adopts tandem UIM motifs. Actually, UIM3 is relatively highly conserved in sequence as compared with UIM1 and UIM2, but the pull-down experiment suggests that it is unlikely to bind with Ub (Supplemental Fig. S3). From NMR titration experiments, we calculated the dissociation constants for the three single UIM motifs (Table 1). As expected, the single UIM1 and UIM2 bind Ub much weaker than AT3-UIM12. Different from the situation of UIM moieties in AT3-UIM12 where UIM2 binds Ub a little stronger than UIM1, these two individual UIMs exhibit almost similar binding affinities. This is possibly the result of association of the two UIM motifs linked by the relatively flexible region and the structural transformation of AT3-UIM12 especially in the UIM2 moiety that favors Ub binding. Thus, the tandem form of UIM motifs improves the binding affinities with Ub as compared with single isolated UIMs. To investigate the linker region that may be critical to the Ub binding, we constructed three mutants of AT3-UIM12 with different linkers (Fig. 1C). Substitution of the linker region (GS- Sub) weakens the binding with Ub considerably, whereas both insertion (GS-Ins) and substitution of the linker region with RAP80 linker (RAP80-Linker) significantly increase its binding affinity with Ub (Table 1). This result indicates that the relatively long linker is beneficial to the Ub binding, possibly due to the increased flexibility of the long linker or formation of a longer a-helix as in RAP80 [19]. The Hydrophobic Residues of AT3-UIM12 Play Dominant Roles in Ub Binding Ub interacts with some Ub-binding domains (such as UBA) mainly through its hydrophobic patch composed of Leu8-Ile44- Val70 [32]. In this study, NMR titration of Ub with AT3-UIM12 has given similar information of the binding surface on the Ub PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13202