Fig. 7: Lysine to threonine mutations near Survivin’s NES and dimer interface reduce tweezer affinity and impair its inhibitory effect on the Survivin–CRM1 interaction.
From: Specific inhibition of the Survivin–CRM1 interaction by peptide-modified molecular tweezers
Titration of 300 μM TW (a) in the cell with 321 µM Survivin120 K90/103T in the syringe. Titration of 100 µM TW-ELTL (b) and TW-ELTLGEFL (c) in the cell with 2.5 mM Survivin120 K90/103T in the syringe. All titrations were performed in PBS, pH 7.4 at 25 °C. Graphs represent one representative example each from three independent experiments (n = 3). The black lines in the bottom panels are the best fit of the data to a one set of sites model. The heat of dilution was subtracted as constant. For thermodynamic data derived from the graphs see SI7. FAM-labeled unmodified tweezer molecule (d), TW-ELTL (e), and TW-ELTLGEFL (f) (0.2 µM) were titrated with either Survivin120 (200 µM, circles) or Survivin120 K90/103T (400 µM, squares). Survivin120 K90/103T showed greatly reduced tweezer affinities (lower curves). d–f Data are presented as mean values ± SD with n = 3 independent experiments. g Pull-down results after immunostaining. GST-Survivin120-WT or GST-Survivin120-K90/103T were incubated with 50 µM respective tweezer molecule or ELTL/ ELTLGEFL peptides w/o tweezer. GST-Survivin120- or GST-Survivin120-K90/103T-loaded beads were mixed with CRM1 and RanQ69L prey proteins as well as dGTP to allow export complex assembly. Proteins in input and bead samples were analyzed via immunoblotting with antibodies specific for CRM1 or GST. WT, wildtype. One representative example of two independent biological replicates is shown. Samples derive from the same experiment and gels/blots were processed in parallel. h Quantification of two independent pull-down experiments. After subtraction of the CRM1 negative control from the pulled CRM1 intensity, the latter is normalized by the GST–Survivin intensity and afterwards normalized by the CRM1 intensity without tweezer incubation. Export complex assembly is only compromised by peptide tweezers in the wildtype Survivin120, but not in the mutant. No ligand: black; TW: light blue; TW-ELTL: blue; TW-ELTLGEFL: dark blue; ELTL peptide: light gray; ELTLGEFL peptide: dark gray. Source data are provided as a Source Data file.
