Fig. 5: Analysis of the swapping activities of SENP5 for SUMO1 and SUMO2 substrates.

a SDS-PAGE of the endpoint assays of SENP5_CD using the RanGAP1-SUMO1 and RanGAP1-SUMO2 wild types, and RanGAP1-SUMO1 E67D and RanGAP1-SUMO2 D63E point mutants as substrates. (Right) Plot of the fraction of the RanGAP1-SUMO1 wild type and point mutants after 30 min reaction. Data values represent the mean ± SEM, n = 3 technical replicates. Significance was measured by a two-tailed unpaired t test relative to wild-type. All data were analyzed with a 95% confidence interval. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Exact P values from left to right: 0.0001 and 0.0173. b Plot of the proteolytic cleavage of SUMO1-Rho, SUMO2-Rho wild types and SUMO1-Rho E67D and SUMO2-Rho D63E point mutants to test the activity of SENP5_CD. The plot is an average of a triplicate experiment. c Plot of a competition assay by measuring the interference of the SENP5_CD activity for SUMO2-Rhodamine substrate by competition with RanGAP1-SUMO1 and RanGAP1-SUMO2 wild types, and RanGAP1-SUMO1 E67D and RanGAP1-SUMO2 D63E point mutants at different concentrations. (Right) Inhibitory constant values (IC₅₀) for RanGAP1-SUMO1 and RanGAP1-SUMO2 wild types, and RanGAP1-SUMO1 E67D and RanGAP1-SUMO2 D63E point mutants substrates on the SENP5_CD wild type activity on SUMO2-Rhodamime fluorogenic substrate. Data values represent the mean ± SEM, n = 3 technical replicates. d Immunoblot analysis of the labeling of endogenous full length human SENP3 in HEK293T lysates with activity-based probes of either SUMO1 or SUMO2 wild type and point mutants. Anti-actin antibody was used as a loading control. n = 3 technical replicates. e Close-up view of the ribbon representation of interfaces of SENP5_CD-SUMO1 wild type depicting the basic patch region around Arg624. Interface residues are labelled and shown in stick representation. f Close-up view of the ribbon representation of interfaces of SENP5 _CD-SUMO1 E67D point mutant depicting the basic patch region around Arg624. g Superposition of the SENP5 structures (blue surface) in complex with SUMO1 wild type (blue ribbon) or SUMO1 E67D (orange ribbon). Source data are provided as a Source Data file.