Extracellular\nPeptide-Ligand Dimerization Actuator\nReceptor Design for Reversible and Spatially Dosed 3D Cell-Material\nCommunication

Transmembrane receptors\nthat endow mammalian cells with the ability\nto sense and respond to biomaterial-bound ligands will prove instrumental\nin bridging the fields of synthetic biology and biomaterials. Materials\nformed with thiol-norbornene chemistry are amenable to thiol-peptide\npatterning, and this study reports the rational design of synthetic\nreceptors that reversibly activate cellular responses based on peptide-ligand\nrecognition. This transmembrane receptor platform, termed Extracellular\nPeptide-ligand Dimerization Actuator (EPDA), consists of stimulatory\nor inhibitory receptor pairs that come together upon extracellular\npeptide dimer binding with corresponding monobody receptors. Intracellularly, <i>Stimulatory EPDAs</i> phosphorylate a substrate that merges\ntwo protein halves, whereas <i>Inhibitory EPDAs</i> revert\nsplit proteins back to their unmerged, inactive state via substrate\ndephosphorylation. To identify ligand-receptor pairs, over 2000 candidate\nmonobodies were built <i>in silico</i> using PETEI, a novel\ncomputational algorithm we developed. The top 30 monobodies based\non predicted peptide binding affinity were tested experimentally,\nand monobodies that induced the highest change in protein merging\n(green fluorescent protein, GFP) were incorporated in the final EPDA\nreceptor design. In soluble form, stimulatory peptides induce intracellular\nGFP merging in a time- and concentration-dependent manner, and varying\nlevels of green fluorescence were observed based on stimulatory and\ninhibitory peptide-ligand dosing. EPDA-programmed cells encapsulated\nin thiol-norbornene hydrogels patterned with stimulatory and inhibitory\ndomains exhibited 3D activation or deactivation based on their location\nwithin peptide-patterned hydrogels. EPDA receptors can recognize a\nmyriad of peptide-ligands bound to 3D materials, can reversibly induce\ncellular responses beyond fluorescence, and are widely applicable\nin biological research and regenerative medicine.