Figure 4: The critical role of the β-grasp domain of SUMO1 on binding and activity enhancement of SENP1.

(a) Left panel, overlap of the ¹H–¹³C-methyl TROSY spectra of SENP1 (red), and SENP1 in complex with SUMO1_1–92 complex (blue). Right panel, overlay of the spectra of wild-type SENP1 (green) and SENP1 (C603S) (red). Stereo-specific assignments of the Val532 methyl groups are indicated. (b) Stereo view of representative SENP1 crystal structures showing Val532 and surrounding residues for relating structural changes to the observed chemical shift changes in the methyl groups of Val532 shown in a. The Val532 γ₂-methyl group faces the His533 aromatic ring while the γ₁-methyl group is pointing away. The PDB IDs for the structures are: red-2IYC (apo-state) and yellow-2IY0 (complex state). (c) Steady-state kinetic measurements for SENP1, free (left panel) and when saturated with SUMO1_1–92 (right), using DUB-Glo as a substrate. The fitted kinetic parameters are shown with the corresponding data. (d) Steady-state kinetic measurements for USP5, free (left panel) and when saturated with ubiquitin_1–71 (right), using DUB-Glo as a substrate. The fitted kinetic parameters are shown with the corresponding data. For c and d, error bars were obtained from triplicated measurements.