Fig. 5
From: Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets
Mammalian display saturation mutagenesis to identify substitutions that improve or reduce binding. a TB1G1:TEAD binding in surface display tested over a dilution series of biotinylated, 6xHis-tagged TEAD + stain (200 nM → 64 pM), and with different staining methods conferring different avidities. Top row: 1-step co-incubation of TEAD and streptavidin for tetravalent staining (4x). Middle row: 1-step co-incubation of TEAD and anti-His antibody for bivalent staining (2x). Bottom row: 2-step incubation, first with TEAD followed by pelleting and resuspending in solution with streptavidin, for monovalent staining (1x). Signal:noise ratios, quantitated by Alexa Fluor 647 levels in transfected (signal, S) vs. untransfected (noise, N) cells using flow cytometry, are at the top of each plot. b Mutational tolerance of TB1G1, evaluated by saturation mutagenesis. Heat map displays enrichment scores for every possible non-Cys substitution in TB1G1, tested for TEAD binding in SDGF surface display screening (20 nM, monovalent staining). Rows are amino acid substitutions, grouped by chemical category. Columns are the TB1G1 protein sequence, duplicated below the heat map. Enrichment score represents a variant’s fold-change in population abundance after two rounds of TEAD sorting versus its input abundance, normalized to TB1G1, and log2-transformed. Warm colors are enriched variants (improved binding); cool colors are depleted. X: Average enrichment score. The TB1G1 sequence and structures are color coded for the average enrichment scores of each residue, with warm colors indicating tolerance to substitution and cool colors indicating intolerance. Asterisks in the heat map indicate mutations combined to create TB1G2. c, d Soluble TB1G2 and TB1G2-W40P were analyzed by RP-HPLC (bottom) and SDS-PAGE (right insets) in either non-reducing (PBS) or reducing (10 mM DTT) conditions. TEAD-binding KD values were 368 ± 4 pM (c, TB1G2; 0.044, 0.133, 0.4, 1.2, and 3.6 nM in singleton) and 3.78 ± 0.05 nM (d, TB1G2-W40P; serial 2-fold dilutions of 50 nM → 390 pM in duplicate) by SPR (left insets; SPR responses in black, binding model fits in red). Residues mutated from TB1G1 are in blue, bold font in the sequences. Please see the Methods and Supplementary Table 4 for SPR methodology and analytical models
