Fig. 3. Ditopic CC ligands with various protein linkers activate cognate receptors.

a Alternative ditopic CC ligands expressed in bacteria are engineered by fusing the N-terminus of a monomeric CC to the C-terminus of another, using various protein linkers. Arrows indicate N- to C- terminus directionality. b Ditopic ligands were expressed in E. coli, after SUMO-tagging. Flexible (SGSSGS) and more rigid (EAAAK) sub-units^58,59 were used to engineer a total of three linkers (l_x) (see “Methods” and Supplementary Table S7) c SEAP activity [U/L] in HEK293T cells transiently transfected with A-type_JAK/STAT receptor with linker α₂ (8 aa, GS repeats). Following transfection, cells were incubated with either 0.12 μM purified A’-A’ dipeptide ligand, with or without a SUMO tag with various linker lengths (l₁: pink bars, l₂: yellow bars, or l₃: light blue bars), 0.12 μM A’-A’ reference ligand (ref.: blue bar; see Fig. 2c), or 0.24 μM A’ monomeric CC (see “Methods”). SEAP expression was monitored to assess receptor activation. Bars indicate mean activity; individual data points represent independent triplicates, performed on the same day. Fold changes are calculated against cells incubated with A’ monomeric CC and are shown above bars (for statistical analysis, see Supplementary Table S4). d HEK293T cells expressing A-type_JAK/STAT receptor with α₂ were incubated with Cy3-labeled SUMO-tagged A’-A’ dipeptide with l₂ (see “Methods”) and imaged with a confocal fluorescent microscope. e Fluorescence confocal micrograph images of HEK293T cells expressing A-type_JAK/STAT receptor with α₂ following incubation with Cy3-labeled SUMO-tagged A’-A’ dipeptide with l₂ (n = 1, see “Methods”). Cells were stained with Hoechst stain (blue). Cy3 in yellow. Scale bar (50 μm) is shown on the bottom-right of the image and intensity bar on top-right. Cy3 excitation: 553 nm, emission: 570–620 nm. Hoechst excitation: 405 nm, emission: 410–450 nm. Source data are provided as a Source Data file.