Figure 5.
Non-hydrolyzable diUb Probes Specifically Target the S1-S2 Site of OTUD2
(A) The isolated OTU domain of OTUD2 (OTUD OTU) at a concentration of 0.25 μM was incubated with 2.5 μM TAMRA-labeled mUb (m), K11-linked, or K48-linked diUb-PA probes for indicated times.
(B) OTUD2 OTU was incubated with TAMRA-labeled mUb-PA or the seven differently linked diUb-PA probes as in (A) for 5 min. OTUD2 OTU coupled to TAMRA-labeled mUb-PA (m) or diUb-PA (d) is indicated. f-Ub(2)-PA indicates the unbound fluorescent (di)Ub probe.
(C) 15 nM OTUD2 OTU was incubated with Ub-AMC or diUb-AMC substrates at different indicated concentrations, and the increase in fluorescence was measured over time (Figure S4A). The initial reaction rates (VI) were calculated to generate the Michaelis-Menten plot.
(D) 0.1 μM full-length (FL) OTUD2 or isolated OTU domains of WT OTUD2 (aa 147–314), an S1 site mutant (aa 147–314, AI200-201DD), an S2 site mutant (aa 147–314, I292Q, V295Q), and the catalytically inactive C160A mutant were incubated with 1 μM TAMRA-labeled K11-linked diUb-PA probes. Samples were taken at indicated time points for SDS-PAGE and subsequent fluorescence scanning. All panels were scanned and processed similarly except panel II, which was scanned separately. See Figure S2B for full SYPRO orange stained gels.
(E) 15 nM of the OTUD2 variants mentioned in (D) were incubated with various concentrations of K11-linked diUb-AMC substrate. The increase in fluorescence was measured over time (Figure S4B). The initial reaction rates (VI) were calculated to generate the Michaelis-Menten plot.
(F) Complex structure of OTUD2 and K11-linked diUb as in Figure 4F (Mevissen et al., 2013).
(G) Close-up of the S1 site interface. Residues Ala200 and Ile201 were mutated in the OTUD2 OTU MutS1 construct.
(H) Close-up image of the S2 site interface. The residues Ile292 and Val295 that interact with the Ub Ile44 patch were substituted in the OTUD2 OTU MutS2 mutant.
Error bars represent the SD of the mean based on duplicate measurements.