E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1

Abstract

OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

Figure 1. E2 enzymes stimulate OTUB1 DUB activity

(a) FRET-based assay for cleavage of internally quenched fluorescent K48 Ub₂ (400 nM) by OTUB1 (30 nM) in the absence and presence of 11 different E2 enzymes (5 µM). (b) The rates of K48 Ub₂ (400 nM) cleavage by OTUB1 (30 nM) are plotted as a function of the log of the concentration of UBCH5B. The data were analyzed and fit using a “log(agonist) vs. response” model to determine EC₅₀. Each rate was measured in triplicate, and error bars represent the SEM for each measurement. (c) Coomassie stained gel showing K48 Ub₂ (15 µM) cleavage by OTUB1 (0.5 µM) in the presence and absence of UBCH5B (25 µM). (d) Steady-state kinetic saturation curve for cleavage of K48 Ub₂ by OTUB1 (30 nM) in the presence (red) and absence (blue) of UBCH5B (10 µM). Each rate was measured in triplicate, and error bars represent the SEM for each measurement.

Figure 2. OTUB1-E2 interactions are required for stimulation of DUB activity

(a) FRET-based assay for cleavage of K48 Ub₂ (400 nM) by OTUB1 (30 nM) and OTUB1^T134R (30 nM) in the presence and absence of UBCH5B (5 µM). Results for OTUB1 +/− UBCH5B are reproduced from Fig. 1a for comparison. (b) Effect of OTUB1 N-terminal deletions on UBCH5B stimulation. The fold-stimulation in the rate of OTUB1 cleavage of K48 Ub₂ (400 nM) is shown for the indicated wild type and OTUB1 N-terminal deletions (30 nM) in the presence of UBCH5B (2 µM). Rates of K48 Ub₂ cleavage in the presence and absence of UBCH5B were measured in triplicate using the FRET-based deubiquitination assay. Error bars represent the SEM for each measurement. (c) Cleavage of ubiquitin-AMC (10 µM) by OTUB1 (5 µM) in the presence and absence of UBCH5B (10 µM).

Figure 3. UBCH5B stabilizes the OTUB1 ubiquitin-binding helix

(a) The structure of hybrid human/worm OTUB1 (green) bound to ubiquitin aldehyde (Distal Ub, orange), and UBCH5B^C85S ~Ub, in which the donor Ub (Proximal Ub, orange) is covalently linked to the active-site serine (Ser85) of UBCH5B (blue) via an oxyester linkage. The zoomed view highlights side chain interactions between the N-terminal helix of OTUB1, the donor ubiquitin bound in the OTUB1 proximal site, and UBCH5B. (b) Effect of mutations in the OTUB1 N-terminal helix on stimulation by UBCH5B. FRET-based assay for the cleavage of K48 Ub₂ (400 nM) by OTUB1 (30 nM) in the presence and absence of UBCH5B (5 µM). (c) Assay as in (b) showing the effect on OTUB1 stimulation by UBCH5B mutations that disrupt contacts with the OTUB1 N-terminus. Results for wild type OTUB1 in panels (b) and (c) are reproduced from Fig. 1a for comparison.

Figure 4. Effect of free ubiquitin and E2 charging on OTUB1 DUB activity

(a) Model for binding of UBCH5~Ub to OTUB1 and K48 Ub₂. The position of the donor ubiquitin was obtained by aligning the structure of UBCH5A~Ub (PDB ID: 4AP4) with UBCH5B in the OTUB1-Ubal-UBCH5B~Ub quaternary complex. (b) FRET-based assay for the cleavage of K48 Ub₂ (400 nM) by OTUB1 (30 nM) in the presence and absence of UBCH5B or UBCH5B~Ub (1 µM) and free ubiquitin (10 µM). (c) Effect of free ubiquitin on OTUB1 activity in the presence of charged and uncharged UBCH5B. The rate of K48 Ub₂ (400 nM) cleavage by OTUB1 (30 nM) as a function of the log of the concentration of UBCH5B (black circles) or UBCH5B~Ub (black squares). Each rate was measured in triplicate, and error bars represent the SEM for each measurement. The were analyzed and fit using a “log(inhibitor) vs. response” model to determine IC₅₀. Curves fit to the data are shown for UBCH5B (red) and UBCH5B~Ub (blue).

Figure 5. E2 enzymes exist in both charged and uncharged states in vivo

(a) Western blot of cell lysates from HeLa and U2OS cells prepared at acidic pH (± β-ME) and probed with anti-UBC13 and anti-UBCH5 antibodies exhibit both charged and uncharged forms of the E2 enzymes. (b) Depiction of three possible states of OTUB1-E2 complexes. Top: OTUB1 (green) bound to K48 Ub₂ (orange) and E2 (blue) or E2 (blue)~Ub (purple) thioester. Bottom: OTUB1 bound to free Ub (yellow) and E2~Ub thioester. The free ubiquitin (yellow) and the ubiquitin (purple) conjugated to the E2 bind in place of K48 Ub₂.