Engineering Synthetic Allostery for Phosphorylation-Based Protein Circuits

Phosphorylation encodes information reversibly and dynamically in cellular signaling pathways. Synthetic protein circuits reprogram cellular behaviors to function as sensors, switches, and amplifiers. In contrast to protease-based designs, kinase-driven phosphorylation promises to enable fast, reversible protein circuits that can sense and respond to endogenous signaling states and environmental stimuli. Current approaches include split-protein reconstitution and domain insertion, which require extensive fine-tuning while the repertoire of engineered kinases is limited. However, mutating negatively charged allosteric hotspots to phosphorylatable residues in co-evolving protein sectors has previously achieved successful rewiring of yeast MAPK pathways. We extend this strategy to expand the protein circuit toolbox by engineering allosterically controlled human kinases. We developed a computational pipeline for statistical coupling analysis to identify sector-connected surface D/E residues in a eukaryotic kinome-wide sequence alignment. We then adapted imaging-based kinase translocation reporters for high-throughput activity profiling using flow cytometry. We conduct an alanine scan to identify functionally-coupled candidate residues for introducing phosphorylation motifs of an input kinase. Our work provides insights into allostery design principles and the systematic development of phosphorylation-based protein circuits with composable engineered kinases.