Fig. 2: Concave 5HCS binder to TGFβRII.

a Left: Design model of 5HCS_TGFBR2_1 (cartoon) binding to TGFβRII (PDB ID: 1KTZ). 5HCS_TGFBR2_1 is colored by Shannon entropy from the site saturation mutagenesis results at each position in blue (low entropy, conserved) to red (high entropy, not conserved). Right: Biolayer interferometry characterization of 5HCS_TGFBR2_1. Biotinylated TGFβRII were loaded to Streptavidin (SA) tips and incubated with 2.7 nM, 0.9 nM, and 0.3 nM of 5HCS_TGFBR2_1 to measure the binding affinity. The binding responses are shown in solid lines and fitted curves are shown in dotted lines. b Circular dichroism spectra from 25 °C to 95 °C for 5HCS_TGFBR2_1. c Crystal structure of 5HCS_TGFBR2_1 in complex with TGFβRII. Left are top and side views of the crystal (blue and gray) superimposed on the design models (green and white). In the middle, TGFβRII is shown in surface view and colored by electrostatic potential (using ChimeraX; red negative, blue positive). On the right, detailed interactions between 5HCS_TGFBR2_1 (blue, green) and TGFβRII (gray, white) are shown. d Heat map of the log enrichments for the 5HCS_TGFBR2_1 SSM library selected with 1.6 nM TGFβRII at representative positions. Enriched mutations are shown in red and depleted in blue. The annotated amino acid in each column indicates the residue from the parent sequence. e Dose-dependent inhibition of TGF-β3 (10 pM) signaling in HEK293 cells. The mean values were calculated from triplicates for the cell signaling inhibition assays measured in parallel, and error bars represent standard deviations. IC₅₀ values were fitted using four-parameter logistic regression by Python scripts. Source data (a, b, e) are provided in the Source Data file.