Fig. 4
From: Kinetic analysis of multistep USP7 mechanism shows critical role for target protein in activity
Development of synthetic p53-derived substrates for USP7. a Schematic domain structure of p53 with trans activation domain (TAD), core region, tetramerization domain (TET) and C-terminal region indicated (CT). Close-up of CT highlights lysines (bold) known to be ubiquitinated39 and the TRAF recognition motif (underlined)29,43. K382 (red) is used as target lysine in the synthetic p53Ub reagents, underneath the sequence coverage of the peptides is indicated. b Three synthetic p53Ub-peptides are generated with different linkage types: covalently binding (vinylamide linkage, (I)), uncleavable (triazole linkage, (II)) and cleavable (native linkage, (III)). c The affinity of USP7 for the covalent-binding p53UbVA increases if the TRAF domain is present. USP7 constructs with and without TRAF domain have been incubated with the probe for 15 min at RT and analysed on a Coomassie gel. d The affinity of Ubl45 for the substrate-bound TCD is similar to CDUb (2.9 µM compared to 0.59 µM, Fig. 3c). Around 50 units of Ubl45 were immobilized on the chip before a titration range of p53Ub-bound TCD was flown over. Their equilibrium binding values were plotted and fitted to get the displayed KD-value, with the SD (±)
