1H-15N HSQC spectra of Ub were obtained and the chemical shift changes were measured for each molar ratio. The sequence- specific backbone assignment for Ub was achieved by comparing the almost identical resonance dispersion of Ub on the 1H-15N HSQC spectra as reported previously [60]. The combined average chemical shift changes (Ddave) were calculated as Ddave = [(0.2*DdN) 2 + (DdHN) 2]1/2, where DdHN and DdN are the chemical shift changes in the 1H and 15N dimensions, respectively. Similarly, the Ddave values of 15N-AT3-UIM12 titrated by Ub diUb were also obtained. The dissociation constant (KD) for the UIM-Ub binding was obtained by fitting the titration curves [61]. For the binding of AT3-UIM12 with Ub, an apparent dissociation constant (KD,app) was obtained by applying a simplified 1:2 fit, provided that the two UIM motifs bind Ub with equal binding affinity. GST Pull-down Assay For pull-down assay, the GST-fused Ub was added to the glutathione Sepharose 4B beads (Amersham Biosciences) in a PBS buffer (10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4), and the suspension was agitated at 4uC for 30 min. The beads were washed three times in the same buffer to remove any unbound protein. An equal molar amount of GB1- fused UIM12 or UIM3 of AT3 (containing a C-terminal His6-tag) was added, and the suspension was agitated at 4uC for about 2 h. The beads were recovered by centrifugation, and, after excessive washing, the sample was re-suspended in the sample buffer and subjected to SDS-PAGE, followed by Coomassie staining or Western blotting with an anti-His antibody. Similar pull-down experiments were carried out between GST-fused AT3 UIMs and diUb or polyUb. The GST-fused AT3-UIM1, -UIM2 or -UIM12 was incubated for 2 h with either K48-linked or K63-linked diUb (8.5 mg, 0.5 mM) or polyUb mixture (2.5 mg) with a chain length ranging from one to seven (Ub1,7, purchased from Boston Biochem Inc.) The pull-down samples were subjected to SDS- PAGE, followed by Western blotting with an anti-Ub antibody. Isothermal Titration Calorimetry The ITC experiments were performed on a MicroCal iTC200 at 25uC in an ITC buffer (25 mM Tris-Cl, 50 mM NaCl, pH 7.5). The GB1-AT3-UIM12 protein (2 mM) stocked in a syringe was injected into a 300-mL sample of linear, K48- or K63-linked diUb with the same buffer. The concentration of diUb sample in the cell was 100 mM as determined by spectrophotometry with a coefficient of 2980 cm21M21 at 280 nm. The binding constants were calculated by fitting the data with one-site, two-site or sequential binding model using Origin 7.0 software (OriginLab Corp.) for ITC. Supporting Information Table S1 Experimental Restraints and Structural Statistics of Free and Ub-Bound Forms of AT3-UIM12 Found at: doi:10.1371/journal.pone.0013202.s001 (0.08 MB PDF) Table S2 Summary of the ITC data for AT3-UIM12 binding with diUb chains by different fitting models Found at: doi:10.1371/journal.pone.0013202.s002 (0.01 MB PDF) Figure S1 1H-15N HSQC Spectra Showing Assignments of Chemical Shifts of the AT3-UIM12 Forms. (A) Free AT3-UIM12 form. The residues are numerated according to the sequence of AT3-UIM/Ub Interaction AT3. (B) Ub-bound AT3-UIM12. The spectrum of Ub-bound form was recorded at the AT3-UIM12/Ub ratio of 1:4. Found at: doi:10.1371/journal.pone.0013202.s003 (0.12 MB PDF) Figure S2 Steady-state hetero-nuclear 15N-{1H}-NOE exper- iments of AT3-UIM12 in the absence (A) and presence (B) of Ub. The spectra on the left were recorded with proton saturation, while those on the right were recorded without proton saturation. The ratios of the peak intensities represents the hetero-nuclear 15N-{1H}-NOE values for the backbone amides. Red, positive NOE; green, negative NOE. Found at: doi:10.1371/journal.pone.0013202.s004 (0.42 MB PDF) Figure S3 Pull-down Experiment Showing Bindings of AT3- UIM12 and AT3-UIM3 with Ub. The GST pull-down experi- ments were carried out between GST-Ub and AT3-UIMs, and detected by Coomassie staining and Western blotting with anti-His antibody. AT3-UIM12 pulled down by GST-Ub is indicated by an asterisk. AT3-UIM12 refers to the tandem UIMs of AT3 (residues 222–263); and AT3-UIM3 denotes the third UIM motif (residues 335–354). Found at: doi:10.1371/journal.pone.0013202.s005 (0.08 MB PDF) Figure S4 NMR Titration Showing the Interaction of Tandem AT3-UIM12 with Ub. (A) Traces for the chemical shift changes of the representative residues of AT3-UIM12 upon Ub titration. The peaks are colored from red (UIM/Ub = 1:0) to coral (1:6). (B) Titration curves for two equivalent residues, Ser236 in UIM1 and Ser256 in UIM2 (left panel), and Ala232 and Ala252 (right), in tandem AT3-UIM12 upon increasing amount of Ub. Note that the Dd values of the residues in UIM2 moiety upon Ub titration reach the plateau with lower amount of Ub than the correspond- ing ones in UIM1. Found at: doi:10.1371/journal.pone.0013202.s006 (0.14 MB PDF) Figure S5 Interaction of the Linker Mutants of AT3-UIM12 with Ub. The titration curves for wild-type AT3-UIM12 and its linker mutants (I240A, I240P, Q238E/E239T) binding with Ub are plotted using the chemical shift changes of Leu71 of Ub as a representative. The dissociation constants for these mutants binding with Ub are listed in Table 1. Found at: doi:10.1371/journal.pone.0013202.s007 (0.03 MB PDF) Figure S6 Pull-down Experiments Showing the Interactions of Tandem UIM12 and Individual UIMs of AT3 with Different polyUb Chains. The Pull-down experiments were performed on various lengths of polyUb (Ub1,7, K48- or K63-linked) with GST-fused UIM1, UIM2 or UIM12. The protein bands were detected by Western blotting with an anti-Ub antibody. Found at: doi:10.1371/journal.pone.0013202.s008 (0.13 MB PDF) Figure S7 Interactions of Tandem AT3-UIM12 with Different Forms of Ub by NMR Titrations. (A) Titration of 15N-labeled AT3-UIM12 with monoUb. (B) Titration of 15N-labeled AT3- UIM12 with linear diUb. (C) Titration of 15N-labeled AT3- UIM12 with K48-linked diUb. With the progress of K48-linked diUb titration, some peaks in the 1H-15N HSQC spectra experience large chemical shift changes and peak broadening. (D) & (E) Titration curves for two representative peaks (Q230 and S236) showing distinct binding affinities for monoUb, linear diUb and K48-linked diUb. PLoS ONE | www.plosone.org 11 October 2010 | Volume 5 | Issue 10 | e13202