Extended Data Fig. 7: Determining the DUB activity, inhibitor sensitivity, and SHMT2 inhibition of structure-guided mutants.
From: Molecular glues that inhibit deubiquitylase activity and inflammatory signaling
a, SDS-PAGE analysis of purified BRISC mutants. Each purified protein sample was generated from one purification. Uncropped gels are in Source Data Extended Fig. 7. b, Activity of BRISC mutants against an IQF di-ubiquitin substrate. Data points are mean ± SEM of three independent experiments carried out in technical duplicate. c, FX-171-C IC50 values from inhibition assays shown in Fig. 4. d, Protected and deprotected peptides from HDX-MS mapped onto the FX-171-C binding site. Peptides are coloured blue to indicate protection and red to indicate deprotection, after incubation with FX-171-C. e, Superimposition of the SHMT2 dimer from BRISC-SHMT2 structure (PDB: 6R8F) onto BRISC-FX-171-C dimer structure. SHMT2 α6 helix clashes with the BLUE binding site. f, Mutated residues in BRCC45 are not in close proximity to the SHMT2 binding site in the BRISC-SHMT2 structure (PDB: 6R8F). g, Spectral Shift (Dianthus) assays measure the binding of SHMT2(A285T) to labelled His-BRISC in the absence and presence of compounds. KD is calculated by plotting the ratio of the fluorescence intensities at 650 nm and 670 nm against SHMT2 concentration, with a GraphPad Prism equation for one-site total binding. Data points are mean ± SEM of three independent experiments.
