Fig. 1

USP7 activation by the C-terminal tail happens in cis. a Schematic domain representation of USP7 and constructs used in this study. Active site residues and domain names are indicated: TRAF TRAF domain, CD Catalytic domain, 1–5 Ubl domains 1 through 5, tail. The activating C-terminal peptide (res. 1083–1102), marked in purple. The graphical representation of the constructs is used in other figures. b Analysis of USP7 constructs on SEC-MALLS shows monomer/dimer equilibrium. CD45 (100 µL of 45 µM) or FL (80 µL of 20 µM) were loaded on a Superdex 200 gel filtration column. Absorbance at 280 nm (dark red: CD45; dark blue: FL) was monitored and eluted peaks were analysed for molecular weight (red: CD45; blue: FL) by in-line MALLS. For CD45 the molecular weights of the monomer (69 kDa) and dimer (138 kDa) are indicated with the dotted line. c Activation of USP7CD by Ubl45 requires the very C-terminal tail. CD (20 nM) was mixed with Ubl45 variants as indicated and tested for DUB activity using UbRho. d USP7CD (20 nM) was incubated with a titration range of Ubl45, and tested in a deubiquitination assay as in 1c. These initial velocities were plotted against the concentration to yield an apparent K_D. Each data point is the mean ± SD of n = 2 measurements. e CD12345^ΔC (20 nM) has similar activity to CD and can be activated by the tail: when titrating in CD12345^C223A, which has no activity, the tail will activate the tailless construct upon dimerization. The activity readout shows that this dimerization-dependent activation of USP7 happens at micromolar concentrations in line with affinity of 1d. Binding of ubiquitin substrate to CD12345^C223A causes an inhibitory effect above 2 µM, therefore only lower concentrations were used to extrapolate a K_D,app (blue dashes). The red dotted lines indicate the activity of WT CD12345 for comparison