Figure 1.
USP13 prefers to bind with Ub.
A, Domain architectures of USP5 and USP13. NT, N-terminal domain; ZnF, zinc-finger domain; UBA, Ub associated domain; C-box and H-box, two separated lobes of the catalytic domain. B, GST pull-down analysis of USP13 (purified with His6 tag from E.coli) with GST-fused Ub and other UbL proteins. 50% USP13 was loaded as a control. The samples were analyzed by SDS-PAGE with Coomassie blue staining.
Figure 2.
USP13 exhibits weak deubiquitinating activity for K63-linked polyUb chains.
A, Hydrolysis of Ub-AMC catalyzed by recombinant GST-tagged USP13 or USP5. Ub-AMC (250 nM) was incubated with USP13 (50 or 500 nM) or USP5 (1.5 nM) or USP13-C345A (500 nM). B, Cellular ubiquitination levels in the presence of various USPs. HEK 293T cells were transfected with vector, FLAG-tagged USP13, USP5 or their variants. USP13-C345A and USP5-C335A are the active-site point mutants. Before harvesting, the cells were incubated with 10 µM MG132 for 10 hrs. Then, the cell lysates were detected by immunoblotting using an anti-Ub antibody. C, Hydrolysis of Ub-AMC catalyzed by FLAG-tagged USP13, USP13-C345A and USP5 purified from HEK 293T cells. Ub-AMC (250 nM) was incubated with USP13 (20 nM), USP13-C345A (20 nM) or USP5 (2 nM). D, Hydrolysis of the Ub tetramer by GST-fused USP13 and its C345A mutant. GST-USP13 and its mutant C345A (2 µM) were incubated with K48- or K63-linked Ub4 (0.1 µg/µL), and the reaction product in each time point was detected by immunoblotting using an anti-Ub antibody. E, As in (D), hydrolysis of the Ub octamer by USP13. GST-USP13 (2 µM) was incubated with K63-linked Ub8 (0.1 µg/µL). GST was set as a negative control.
Figure 3.
USP13-ZnF cannot bind with Ub but USP5-ZnF can.
A, Sequence alignment of the ZnF domains from different USPs. The alignment was performed with ClustalX. The secondary structures of USP13-ZnF are indicated with α-helices in blue box and β-strands in orange arrows. The most important residues for Ub binding are marked in purple for outside Loop 2 or in green for inside Loop 2. B, ITC profiles of USP13-ZnF and USP5-ZnF with Ub. The Ub sample (1 mM stock) was injected into buffer (left), USP13-ZnF (middle) or USP5-ZnF (right) in 25 injections with 8 µL each. The concentrations of USP13-ZnF and USP5-ZnF are 50 µM.
Figure 4.
Structure determination of USP13-ZnF and comparison with USP5-ZnF.
A, Superposition of the backbone traces of 10 representative structures. B, Ribbon diagram of a representative structure of USP13-ZnF with secondary structure elements and a zinc ion labeled. The α-helices are colored in red, the β-strands were in yellow, and the loops were in green and the zinc atom is in blue. C, Comparison of the structures of USP13-ZnF (lemon) with USP5-ZnF (pink). The L2 loop between β2 and α1, which is not well superposed, is marked in gray. D, Electrostatic surface of the USP5-ZnF domain from X-ray crystallography (PDB ID: 2G45). The positive charges are shown in blue and the negative charges are in red. The Ub tail, LRLRGG, is represented by a ribbon (orange) on the structure, and the key residues for the interaction are also labeled on the surface. E, Electrostatic surface of the USP13-ZnF domain by NMR analysis. Shown is in the same orientation with in (D). The corresponding residues (arrow indicated) in USP5-ZnF and USP13-ZnF responsible for the interactions are also labeled on the surface. All the structures were generated and displayed by PyMol. F, Overlay of the 1H-15N HSQC spectra of USP5-ZnF in the absence (red) and presence (cyan) of Ub (molar ratio, 1: 0.5). G, Plot of the relative peak heights of amides against the residue number of USP5-ZnF. The peak intensities were normalized to those without addition of Ub. The bars in negative denote prolines or unassigned residues. The solid and dashed lines indicate the values of Mean and Mean-SD for the changes of peak heights.
Table 1.
Dissociation constants of wild-type USP5-ZnF and its mutants binding with free Ub.
Figure 5.
Gain of the catalytic activation of USP13 through ZnF-domain swapping from USP5.
A, Non-activating catalysis of USP13 by free Ub. Ub-AMC (250 nM) was incubated with GST-USP13 (1 µM) for 100 s, and then Ub (38 µM) was added into the reaction mixture. B, Activating catalysis of USP5 by free Ub. In comparison with wild-type USP5 that exhibits high Ub activation, the USP5 mutant (R221K/R222W/Y223F) loses the property of being activated by Ub. Ub-AMC (250 nM) was incubated with USP5 or its mutant (10 nM) for 50 s, and then Ub (100 nM) was added into the reaction mixture. C, Schematic diagram of the domain-swapped mutant of USP13. USP13-5ZnF denotes a chimera enzyme of USP13 with a substituted ZnF domain from USP5. D, USP13-5ZnF can be activated by free Ub. Ub-AMC (250 nM) was incubated with USP13-5ZnF (100 nM) for 50 s, and then Ub (1 µM) was added into the reaction mixture. All the reactions were monitored by fluorescence of released AMC at 460 nm.
Figure 6.
Solution structure of the tandem UBA12 domain of USP13.
A, Sequence alignment of the two UBA domains of USP13 with some classical UBA domains. The alignment was performed with ClustalX. The pink boxes denote α-helices, and the MGF motif is conserved in all UBAs. B, Ensemble of 5 structures superimposed on the polypeptide backbones of UBA1 or UBA2. The graphs were generated by using MOLMOL. C, Ribbon representation of the solution structures of UBA1 and UBA2, and their comparison. The structures were generated and displayed by PyMol.
Figure 7.
Binding of USP13-UBA12 with various types of diUb chains.
A, NMR titration for mapping the binding surface on USP13-UBA12. Shown is the overlay of 1H-15N HSQC spectra of USP13-UBA12 in the absence (blue) or presence of increasing amounts of Ub (gray, 1: 0.6; orange, 1: 1). The residues on the classical binding patch are labeled in the spectra. B, ITC profiles of USP13-UBA12 with various types of diUb or monoUb. The UBA12 samples (1.25 mM stock) were injected into K63-, K48-, linear diUb (0.1 mM) or monoUb (0.2 mM) in 25 injections with 1.5 µL each.
Figure 8.
USP13 regulates cellular levels of CD3δ.
A, Dose-dependent experiments for USP13 or USP5 and their mutants on the CD3δ levels. HEK 293T cells were co-transfected with equal amount (1 µg) of CD3δ-HA and increasing amount (0.5, 0.75 and 1.0 µg) of FLAG-tagged USP13, USP13-C345A, USP5 or USP5-C345A. Cells were harvested and lysed 36 hrs after transfection, and then the samples were analyzed by Western blotting. Data analysis (n = 3) for the amount of CD3δ versus amount of the transfected DNA of USP13, USP5 or their mutants using western blotting and quantified by the intensity of western blotting. B, Effects of the UBA12 mutant or ZnF-deletion mutant of USP13 on the CD3δ levels. USP13-UBA12M is the mutant with two point mutations in each UBA domain (M664E/M739E). USP13-ΔZnF is the mutant with deletion of the ZnF domain. Data analysis was performed as indicated in A.