Fig. 5
From: Kinetic analysis of multistep USP7 mechanism shows critical role for target protein in activity
The p53-derived tools stress the importance of both the TRAF and Ubl45 domain. a Binding of the TAMRA-labelled p53 peptide is determined by incubating 25 nM of TAMRAp53 with a dilution series of various USP7 constructs in FP assays. The obtained KD values for each USP7 construct are stated and indicate that in presence of the TRAF domain, the Ubl12345 domain affects recognition. b The non-hydrolysable p53Ub construct acts as an inhibitor for USP7 in deubiquitination assays. USP7 constructs, corrected for their activity (CD12345: 0.35 nM, TCD: 200 nM, CD: 75 nM, USP7FL: 0.35 nM) were incubated with increasing amounts of p53Ubinh and analysed for activity in a deubiquitination assay using a single concentration of UbRho. The raw data indicate that both the TRAF domain and the C-terminal Ubl domains increase the affinity towards the p53 construct. For clarity’s sake a limited number of concentrations is shown. c The observed activity for USP7 constructs after incubation with p53Ubinh (see B), is plotted against the concentration of inhibitor used. Fitting yielded IC50 values and standard deviation as shown. The data points for a, b are the mean ± SD of at least n = 2 experiments. All reported values include the SD (±)
