(A) BINDI (gray) was docked to the hydrophobic binding groove of Bcl-xL (red) by alignment to a bound BH3 peptide (not shown). The docked pose undergoes successive rounds of design with ROSETTA, in which BINDI residues within 8 Å (Cβ atoms indicated by yellow spheres) and then 12 Å (cyan spheres) of the interface are available for computational mutation, while hotspot residues (pink spheres) are held constant. (B) Hotspots (pink) are borrowed from native BH3 motifs or designed peptides, transferred to the BINDI scaffold and restricted from mutation during design (termed ‘side chain grafting’). (C) A novel motif-grafting method samples all possible docked configurations of the BINDI scaffold (multiple colors) in the Bcl-w (green) binding groove. (D) The docked position of Bcl-w-targeting design W-CDP03 (green cartoon) is shifted one helical turn relative to (E) BINDI (gray cartoon) and exhibits better shape complementarity to Bcl-w (green surface). (F) Selected computationally designed proteins (CDPs) are qualitatively compared with single-concentration BLI assays. Design M-CDP02 is partially specific for Mcl-1, and M-CDP04 is highly specific. (G) Binding constants (KD) are accurately determined with multiple-concentration BLI titrations. Biotinylated Mcl-1 was immobilized to a streptavidin-coated sensor and incubated with the indicated concentrations of soluble αMCL1. Raw data are purple, fitted curves are black. (H) KD values of CDPs, intermediate ECMs, and Bim BH3 (as a fusion to maltose binding protein) were determined with BLI (mean ± SD; n = 3), and binding constants of competing small molecule and peptide inhibitors (citations noted below). (I) Computationally designed proteins (all gray, except M-CDP04/αMCL1 in black which required no further optimization) and their optimized successors (ECMs dashed; better variants solid black) were denatured with guanidinium hydrochloride. The CD signal at 222 nM was measured and loss of signal used to calculate the fraction folded. Mean CD signal ± SD from three scans. (1) Souers et al., 2013; (2) Leverson et al., 2015; (3) Leverson et al., 2015a; (4) Tse et al., 2008; (5) Dutta et al., 2010; (6) Foight et al., 2014; (7) Dutta et al., 2013.