Fig. 4
From: Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets
Mammalian surface display screening identifies TEAD-binding optides. a Scheme for the screening strategy. FasL-TM: transmembrane domain of the FasL protein. b Flow profiles (GFP vs TEAD-streptavidin-Alexa Fluor 647) of the library of designed optides in SDGF; shown are the profiles of the unsorted input library (top left), and the library after one (top right), two (bottom left), or three (bottom right) TEAD sorts. Arrowhead in bottom right corresponds to TB1G1 clone within enriched polyclonal pool. c YAP:TEAD structure from PDB ID 3KYS, focusing on Interface 2. d, e TB1G1 d and TB2G1 e modeled at the same location on TEAD. Both share a conserved “LXXLF” motif with YAP. f, g The TEAD-binding abilities of 293F cells transfected with SDGF-TB1G1 f or SDGF-TB2G1 g and interface mutations are shown. 6x Cys to Ser (6CS) to eliminate disulfide bonds were also tested. Binding quantitated by Alexa Fluor 647 levels in transfected cells using flow cytometry. Shown are the normalized median ± 95% confidence intervals from one experiment. h Soluble TB1G1 is monodisperse in solution by reversed-phase (RP) HPLC (bottom) and SDS-PAGE (right inset). Reduction with 10 mM DTT was complete and demonstrates a mobility shift in both RP-HPLC and SDS-PAGE. TEAD-binding equilibrium binding constant (KD) was 31 ± 2 nM by SPR (left inset, serial 2-fold dilutions of 2 μM → 0.12 nM in duplicate; RU within blue mark used for curve fit in Supplementary Fig. 8a; see Methods and Supplementary Table 4 for SPR methodology). NR: Non-reduced. R: Reduced with 10 mM DTT. i TB1G1 and the poorly-binding F38A variant were tested for inhibition of FLAG-YAP co-immunoprecipition with Myc-TEAD in 293T cell lysate. j, k Dilution series of TB1G1 in the FLAG-YAP:Myc-TEAD co-immunoprecipitation assay. j is one representative blot; k is the YAP:TEAD signal ratio, average ± s.d. (N = 4 lanes per dilution from one experiment), quantitated by band densitometry
