AT3-UIM/Ub Interaction Figure 4. Interaction of Tandem AT3-UIM12 with Ub. A, Diagram of the chemical shift changes (Dd) of AT3-UIM12 against residue number at an AT3-UIM12/Ub molar ratio of 1:2. B, Titration curves for two equivalent residues (Leu229 in UIM1 and Leu249 in UIM2) in tandem AT3-UIM12 upon Ub binding. Note that the Dd value of Leu249 in UIM2 upon Ub titration reaches the plateau with lower amount of Ub than the corresponding Leu229 in UIM1. C, Diagram of the chemical shift changes (Dd) of Ub against residue number at an Ub/AT3-UIM12 molar ratio of 1:2. D, Titration curves for three residues (Ile13, Lys48 and Leu71) in Ub upon GB1-tagged AT3-UIM12 binding. The dissociation constant for the binding of AT3- UIM12 with Ub is listed in Table 1. The solid and dashed lines (A & C) indicate the threshold values of mean and mean plus SD for the chemical shift changes. doi:10.1371/journal.pone.0013202.g004 structure [33] centered by Leu8, Ile44 and Val70 (Fig. 5A). This canonical binding surface has been reported in several related literatures [18,34,35,36,37]. We also obtained direct evidence for the specific binding sites on AT3-UIM12 from intermolecular NOEs. Based on the filtered/ edited-NOESY spectra recorded on the AT3-UIM12/Ub com- plex (at a molar ratio of 1:2), several intermolecular NOE peaks were unambiguously identified. We found that eight residues of AT3-UIM12 have direct contacts with Ub (Fig. 5B). Among these residues interacting with Ub, Leu229, Ala232 and Ser236 are located in the first UIM motif, while their equivalent residues Leu249, Ala252 and Ser256 plus two other residues Arg250 and Ile253 are in the second UIM (Fig. 5C). Definitely, hydrophobic interactions are dominant in the UIM12-Ub complex, for the Leu, Ala and Ile residues constitute the hydrophobic surface on AT3- UIM12. Ser236 and Ser256 may participate in the binding through hydrogen bonds with the backbone of Gly47 in Ub [18]. There are two similar patches but opposite directions on the binding surfaces of AT3-UIM12, each corresponding to one UIM motif and binding with one Ub molecule (Fig. 5D). Interestingly, there is no intermolecular NOE peak observed for the linker residues Gln238, Glu239 and Ile240, suggesting that these residues are not directly involved in the specific binding. Mutation experiments (I240P and I240A) indicate that these residues have limited effect on the binding affinity. However, the double mutant (Q238E/E239T) of AT3-UIM12 gives a similar binding affinity with individual UIMs, suggesting that double mutation of the linker abolishes the association between the two UIMs (Table 1 and Supplemental Fig. S5). Hence, we presume that the chemical shift perturbations observed in the titration experiments (Fig. 4A) might be caused by local conformational change of AT3-UIM12 upon Ub binding. Interactions of AT3-UIM12 with diUb or polyUb Chains Since the NMR investigations provided evidence that the tandem AT3-UIM12 binds with monoUb more efficiently than PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13202