Fig. 3

Affinity of CD for Ubl45 increases with Ub present and is dependent on C-terminal tail. a SPR binding results indicate a weak affinity of USP7CD for either Ubl45 or Ubl45^ΔC. CD was immobilized through GST on the chip and tested for binding with Ubl45 or Ubl45^ΔC. Equilibrium binding values were plotted against concentration and fitted to get an estimated K_D. Responses were normalised using B_max and standard deviation for resulting values is given. b The increased binding between Ubl45 and CDUb depends on the C-terminal tail. Ubl45 or Ubl45^ΔC was immobilized on the chip and the covalent CDUb complex was flown over. A fit was made using the equilibrium binding values yielding a K_D of 590 nM for Ubl45, whereas no binding could be observed for Ubl45^ΔC. Normalisation was carried out using B_max. c Comparison of the affinity of Ubl45 for CD or CDUb shows a remarkable increase. Curves from A and B are replotted to exemplify the change in K_D. d The C-terminal tail was immobilized using biotin and CD or CDUb was flown over to confirm that the tail interacts with the transition state (CDUb) only and not the apo CD. e Overview of affinities between Ubl45 and CD show that Ub enhances the binding of the tail. The values for the upper two rows are determined using SPR, see a–d. The values in the last row have been derived from activity assays, see Fig. 1d and³⁶ for the estimated affinity of the C-terminal tail (*). N.A. not applicable; N.D. No binding detected. f The presence of the Ubl45 domain is essential for increased affinity of CD for ubiquitin, but not the C-terminal tail. The affinity for ubiquitin was measured in an FP assay where TAMRA-labelled ubiquitin was incubated with various USP7 constructs. g Steady-state kinetics analysis of USP7 constructs indicates that the C-terminal tail mainly affects the catalytic rate, while the presence of Ubl45 without the tail enhances the affinity for the ubiquitin substrate. The Michaelis-Menten constant (K_M) and k_cat were obtained by fitting the initial velocity data for various concentrations of UbRho. For a–e, obtained values are displayed ± SD after fitting. For f–g data points are the mean ± SD of n = 2 measurements