Table 1. Apparent Dissociation Constants for the Ub Binding Affinities of Tandem AT3-UIM12, Single AT3-UIM Motifs and Their Mutants. UIM Peptide AT3-UIM12 (WT) AT3-UIM1 AT3-UIM2 AT3-UIM3 AT3-UIM12-I240A AT3-UIM12-I240P AT3-UIM12-Q238E/E239T GS-Subc GS-Ins RAP80-Linker KD,app (mM)a 97.0616.9b 215632 309630 . 3000 14169 17563 333623 195617 16.868.3 39.0611.5 aThe dissociation constants were determined from NMR titration of different GB1-tagged UIM peptides to 15N-labeled Ub. For comparison, we calculated the apparent KD,app values for AT3-UIM12, provided that the two UIM motifs bind Ub equally. bThe chemical shift changes of three individual residues I13, K48 and L71 on Ub were used to calculate the dissociation constants. Data are presented as mean 6 SD. cThe sequences of the linker regions for wild-type AT3-UIM12 and its mutants. WT: 239EIDMED244; GS-Sub: GSSGGS; GS-Ins: EIDGSSGGSMED; RAP80-Linker: EAREVNNQEEK. doi:10.1371/journal.pone.0013202.t001 single UIMs, we further examined the binding properties of single UIMs and tandem UIM12 with diUb or polyUb by GST pull- down experiments. The data show that AT3-UIM12 can pull down both K48- and K63-linked diUb more efficiently than the single UIM1 or UIM2 (Fig. 6). Moreover, AT3-UIM12 can bind with the polyUb mixture more strongly than the single UIM1 or UIM2, no matter whether the polyUb chains are linked by K48 or K63 residue (Supplemental Fig. S6). On the other hand, AT3- UIM12 binds with longer Ub chains more efficiently, which is consistent with the previous reports that AT3 preferentially binds with longer Ub chains [38,39]. To further quantify the binding affinities of AT3-UIM12 with various types of diUb, we performed ITC and NMR experiments. The data for each ITC titration are well-fitted to a sequential two- site binding model rather than a simple one-site or two-site mode (Fig. 7A-C and Supplemental Table S2). Each titration gives two distinct dissociation constants, suggesting that the binding includes two sites on different AT3-UIM12 conformers, one is weak and the other is relatively strong. The results also indicate that the K48- and K63-linked diUb (KD1 = ,10 mM, KD2 = 100,300 mM) bind AT3-UIM12 with similar affinities, but much more strongly than the linear diUb (KD1 = 59 mM, KD2 = 2469 mM). This implies that the strong site on AT3-UIM12 may originate from the allosteric effect during diUb binding. Similar results are also obtained from NMR titrations of AT3-UIM12 with different diUb types, respectively (Supplemental Fig. S7). The binding of AT3- UIM12 with K48-linked diUb is relatively strong, then with linear diUb, and that with monoUb is weak. This suggests that AT3- UIM12 binds different diUb types with different orientations, adaptable to the isopeptide-linked types but inadaptable to the linear one. We also determined the binding affinities of the three linker variants with K48-linked diUb by ITC (Fig. 7D-F and Supple- mental Table S2). As expected, the three mutants of AT3-UIM12 in linker region bind with diUb stronger than with monoUb. AT3-UIM/Ub Interaction Different from those for wild-type AT3-UIM12 (Fig. 7B), the titration data for GS-Sub (Fig. 7D) and GS-Ins (Fig. 7E) cannot be well-fitted to a sequential two-site binding model but can be fitted to a conventional two-site binding model, indicating that alteration of the linker may interrupt the association of two UIM helices binding with diUb. This suggests that the linker region in AT3- UIM12 plays an important role in the association of the two UIM helices. Thus, our biochemical studies reveal that the binding affinity of tandem AT3-UIM12 for polyUb is far larger than those of the individual UIMs, and the linker region in AT3-UIM12 is conducive to structural transformation that may be preferential for the configuration of isopeptide-linked diUb. Discussion More and more multiple copies of domain or motif are found in functional proteins, like the tandem WW domain in HYPA/ FBP11 [40,41], the double SH3 domain in p47phox [31], the two UbL domains in ISG15 [42,43], and the tandem UBA or UIM domains in several proteins related to Ub-proteasome system [3,32,44]. The presence of multiple ligand-binding sites within a protein affords opportunities for multiple simultaneous interac- tions, and is then able to significantly enhance or reduce the efficiency of ligand binding by an allosteric and cooperative (positively or negatively) manner, as compared with the binding to a single domain or motif. As to the multi-UIM-containing proteins, the mutations within a single UIM may produce larger effects than expected if these UIMs function in a non-redundant fashion [5,7]. Furthermore, the relatively modest affinity of single UIM for monoUb leaves open the possibility that the mechanism of high-affinity binding may involve cooperation between multiple UIMs [14,15,45] and polyUb chains [46,47]. However, the previous studies on tandem UIMs from S5a and Vps27p did not find any obvious cooperation for Ub binding [16,17,18]. The UIM portions in both proteins form typical a-helices and interact with Ub directly and individually. On the other hand, some tandem motifs or domains, though non-cooperatively, can mediate formation of a complex or oligomer by binding with polyUb [47,48]. The S5a protein includes another short a-helix in the linker region, however, this helix does not participate in the Ub binding and binding cooperation [18]. The linker region of Vps27p is random and non-structural, and has also been proved not to contribute to the interactions between UIMs and Ub [16]. The interactions of the two UIMs in S5a and Vps27p with Ub have been thought to be independent and non-cooperative. These observations thus argue against a cooperative binding mechanism in the tandem UIMs binding with Ub. The sequence alignment of several tandem UIMs (Fig. 1B) displays high homology in UIM motifs from diverse proteins, but the sequences and lengths of the linker regions between two UIMs are largely different. There are about 20,50 residues inserted in between two UIMs of S5a and Vps27, but the linker region between the two UIMs of AT3 is very short. These adjoining UIM motifs in AT3 raise a possible cooperativity for binding with Ub. Our structure determination and titration experiments reveal that the individual UIMs of AT3 can bind Ub respectively but with lower affinities. The KD values for AT3-UIM1 (215 mM) and AT3- UIM2 (309 mM) are comparable with those for S5a (,350 mM for UIM1 and 73 mM for UIM2) and Vps27p (277 mM for UIM1 and 117 mM for UIM2). When the two UIMs are juxtaposed to interact with Ub, the binding affinity of tandem UIM12, especially the UIM2 moiety, is increased obviously with an apparent KD value of 97.0 mM. As to the diUb or polyUb binding, the binding PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13202