AT3-UIM/Ub Interaction Figure 5. AT3-UIM12 Binds with Ub Through Hydrophobic Interactions. A, Mapping of the significantly perturbed residues by AT3-UIM12 interaction on Ub surface. The residues with chemical shift changes larger than the average have been labeled in pink and those larger than the average plus SD in red (ref. Fig. 3C). The Ub structure is referenced from the crystal structure (PDB code: 1UBQ). B, Intermolecular NOEs observed in the AT3-UIM12-Ub complex. A 1:2 molar ratio of labeled AT3-UIM12 to unlabeled Ub were mixed for 3D 13C-filtered/15N-edited NOESY spectrum. The figure shows the NOE peaks projected onto the 2D plane. C, Mapping of the residues that have the intermolecular NOEs with Ub on the ribbon diagram of AT3-UIM12. D, Mapping of the residues that have the intermolecular NOEs with Ub on the surface diagram of AT3-UIM12, showing two patches for binding with two Ub molecules. The ribbon and surface representations were generated by MOLMOL. doi:10.1371/journal.pone.0013202.g005 affinity of AT3-UIM12 with polyUb is dramatically increased when UIM1 and UIM2 are linked together (Fig. 6 and Supplemental Fig. S6). It seems that the cooperative interaction of AT3-UIM12 with diUb or polyUb is more remarkable than that with monoUb (Supplemental Fig. S7). Although extending the linker significantly increases the binding affinity of AT3-UIM12 with monoUb (Table 1), it may abolish its cooperative interaction with diUb (Fig. 7). Therefore, the altered affinities of AT3-UIM12 for monoUb or diUb may result from structural transformation of the linker region. The backbone dynamics provides further evidence for the conformational change in the linker region, which possibly allows cooperative interaction with Ub. The length of the linker region in tandem UIM motifs is important for a protein to bind with Ub chains, including mono-, poly- and multiUb. In S5a and Vps27p, the long loop linker affords them to bind with any types of Ub but generally weakly and non-cooperatively. The two or three helical portions in the tandem UIM motifs have their respective orientations; thus they bind Ub with different directions. Binding of the individual UIM motif with Ub has no relation to each other and is independently and non-cooperatively (Fig. 8A). Interestingly, although RAP80 contains a rather short linker (7 residues as referred and ,11 as aligned in Fig. 1), the two UIM helices merge together with the linker into a continuous long a-helix when bound with K63-linked diUb [19,46]. Each helical portion binds independently to a Ub unit of K63-linked diUb (Fig. 8B). This binding mode, though efficient and specific for K63-linked diUb (K63-specific avidity), may be not adaptable to the binding of K48-linked or linear diUb, due to the steric hindrance. Because the long helical linker is rather rigid during ligand binding, the tandem UIMs of RAP80 can only specifically bind to K63-linked diUb. However, in AT3, although the two UIM helical portions are less orientated in solution due to PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13202