Peptide-Ligand Responsive Receptors that Enable 3D Cell-Material Communication

Introduction: Transmembrane receptors that endow mammalian cells with the ability to sense and respond to biomaterial-bound ligands will prove instrumental in bridging the fields of synthetic biology and biomaterials. Materials formed with thiol-norbornene chemistry are amenable to thiol-peptide patterning, and this study reports the rational design of synthetic receptors that reversibly activate cellular responses based on peptide-ligand recognition. This transmembrane receptor platform, termed Extracellular Peptide-ligand Dimerization Actuator (EPDA), consists of stimulatory or inhibitory receptor pairs that come together upon extracellular peptide dimer binding with corresponding monobody receptors. Intracellularly, Stimulatory EPDA phosphorylates a substrate that merges two protein halves, whereas Inhibitory EPDA reverts split proteins back to their unmerged, inactive state via substrate dephosphorylation. To identify ligand-receptor pairs, over 2,000 candidate monobodies were built in silico using PETEI, a novel computational algorithm we developed. The top 30 monobodies based on predicted peptide binding affinity were tested experimentally, and monobodies that induced the highest change in protein merging (green fluorescent protein, GFP) were incorporated in the final EPDA receptor design. In soluble form, stimulatory peptides induce intracellular GFP merging in a time and concentration-dependent manner, and varying levels of green fluorescence were observed based on stimulatory and inhibitory peptide-ligand dosing. EPDA-programmed cells encapsulated in thiol-norbornene hydrogels patterned with stimulatory and inhibitory domains exhibited 3D activation or deactivation based on their location within peptide-patterned hydrogels. EPDA receptors can recognize a myriad of peptide-ligands bound to 3D materials, can reversibly induce cellular responses beyond fluorescence, and are widely applicable in biological research and regenerative medicine.

Materials and

Methods: Identifying peptide-ligand monobody-receptor interactions in silico: FLAG, HA, and MYC are well-characterized epitopes with potential to bind to monobodies, and candidate monobodies were identified using a PETEI (Proteins Designed with Targeted Exceptional Interactions) algorithm by altering the three binding loop domains found on protein data bank file 1TTG35. CHARMM (Chemistry at Harvard Macromolecular Mechanics) and Rosetta force fields were then used to identify ten monobodies with the lowest binding energies to FLAG, HA, and MYC ligand-peptides. Identifying monobody receptors in vitro: HA-HA, FLAG-FLAG, and MYC-MYC homodimer peptides (100 µM) were exposed to HEK293 (human endothelial kidney) cells transfected with EPDA receptors containing corresponding monobody receptors bound to zipper, kinase, and phosphatase domains, respectively. Peptide-receptor interactions were evaluated by change in intracellular green fluorescence using flow cytometry (> 10,000 cells/group). EPDA receptor design: the extracellular domain of the zipper, kinase, and phosphatase receptors consist of monobodies specific to HA, FLAG, and MYC, respectively, and the intracellular domains are comprised of a leucine zipper, a phosphoryl group, and phosphatases, respectively. Hydrogel synthesis: EPDA-transfected cells were encapsulated in thiol-norbornene hydrogels formed by mixing (1E6 cells/ml) with norbornene-modified hyaluronic acid (HyaNor, 3 wt%), di-thiol crosslinker peptide (3 mM), and photoinitiator (I2959, 0.05 wt%), and irradiated with UV light (5 min, 6 mW/cm2). Stimulatory and inhibitory peptide photopatterning: unreacted norbornenes in HyaNor hydrogels were used to photopattern thiolated (cysteine, ‘c’) stimulatory HA-c-FLAG and inhibitory HA-c-MYC peptides. Peptide-receptor interactions within 3D hydrogels were assessed via spatial differences in green fluorescence of transfected cells.

Results, Conclusions, and Discussions: Results &

Discussion:
EPDA receptor framework: Peptide ligand heterodimers containing stimulatory HA and FLAG epitopes or inhibitory HA and MYC epitopes were produced using a peptide synthesizer. In the stimulatory construct, HA-FLAG peptides dimerize HA and FLAG receptors (Figure 1A). Internally, a leucine zipper recruits a tyrosine substrate (Tyr) (Figure 1B) that is phosphorylated by an ABL-I tyrosine kinase (Figure 1C). An SH2 domain then attaches to the phosphorylated Tyr (Figure 1D), combining split green fluorescent halves, resulting in cellular activation (Figure 1E). In the inhibitory construct, HA-MYC peptides dimerize the HA and MYC receptors (Figure 1F). Intracellularly, the SH2-Tyr complex is brought towards a phosphatase (1G), resulting in the removal of the phosphoryl group (1H) and separation of the split fluorescent molecule (Figure 1J).
2. Identifying monobody receptors for corresponding peptide-ligands: Computational modeling of predicted HA, FLAG, and MYC epitope-monobody binding energies identified 10 monobodies for each epitope which were tested experimentally (Figure 2). 40% of HA (Figure 2A-B), 50% of FLAG (Figure 2C-D), and 10% of MYC (Figure 2E-F) of these monobodies showed a significant increase in peptide-ligand induced platform activation, highlighting the translatability of our in silico receptor design.
3. Spatial control over ligand-EPDA receptor interactions in 3D hydrogels: Cells were transfected with stimulatory and inhibitory EPDA receptor constructs and encapsulated in 3D HyaNor gels (Figure 3A). Hydrogels were then photopatterned with stimulatory (HA-c-FLAG, 233µm x 233µm squares) or inhibitory (HA-c-MYC, 150µm diameter circles) (Figure 3B-C). Encapsulated cells exhibited minimal platform activation within inhibitory and no peptide regions, and cells within stimulatory regions displayed increased fluorescence, except in areas that also overlapped with inhibitory circles (Figure 3D-G). These results show that EPDA-programmed cells respond to stimulatory and inhibitory peptide-ligands in 3D patterned materials.

Conclusions: By substituting split GFP with a split dCas9 transcriptional activator, peptide-ligands can regulate transcription of cells encapsulated in 3D materials with spatial and temporal control. This novel platform merges the fields of biomaterials and synthetic biology and opens exciting avenues of research including the development of ‘living’ materials and next-generation immunotherapies capable of regulating therapeutic outputs during cell therapy.

Acknowledgements (Optional): The authors would like to acknowledge Dr. Mehdi Benmassaoud for his contributions to this work and funding (NSF EAGER 2037055; NIH NIGMS MIRA R35GM138220).