Fig. 1: Staple scanning of BAD BH3 yields a lead BCL-2 interactor.
a Structure of BCL-2ΔLΔC G101V (PDB 6O0K), highlighting the location of the G101V mutation (red) that shifts E152 (orange) and thereby impairs venetoclax engagement of the canonical groove. Mutation of F104 (blue), which lies in the immediate vicinity of G101V, likewise causes venetoclax resistance29. b Staple scanning library of the BAD BH3 sequence, where “X” represents the inserted S-pentenyl alanine residues that form the i, i + 4 staples and amino acid “B” represents norleucine. For the peptide nomenclature, “4” is an abbreviation for the i, i + 4 staple and the number indicates the location of the first (N-terminal) stapling residue. c Comparative streptavidin pull-downs of biotinylated BAD SAHBs incubated with lysate from HEK 293T cells transiently transfected with full-length, FLAG-tagged, WT BCL-2, as evaluated by anti-FLAG western analysis. Asterisks denote the most effective BCL-2-binding BAD SAHBs. The experiment was performed in biological triplicate using independent preparations of lysates and reagents with similar results. d Helical wheel depiction of the BAD BH3 helix, highlighting the location of three lead i, i + 4 staple positions at the amphipathic interface and extending into the hydrophobic groove. Z represents the insertion position of an S-pentenyl alanine residue N- or C-terminal to the native template sequence. e Streptavidin pull-down of biotinylated BAD SAHBs upon exposure to equimolar recombinant BCL-2ΔLΔC, BCL-XLΔC (aa 1–212), and GST-MCL-1ΔNΔC (aa 170–327) proteins (1 µM), as visualized by electrophoresis and silver stain. The experiment was performed in biological triplicate using independent preparations of proteins and reagents with similar results. f Competitive FP of the FITC-BIM BH3 (3 nM) interaction with BCL-2ΔLΔC (8.6 nM) by BAD SAHBs 4.2, 4.6, and 4.9. Data are mean ± S.D. for experiments performed in technical quadruplicate and repeated three times with independent preparations of proteins and reagents with similar results. Source data are provided as a Source Data file.
