Design and optimization of a kinase-controlled allosteric switch

Post-translational control enables rapid and precise regulation of cell behavior. Despite these advantages, general strategies to build phosphorylation-based synthetic circuits are limited. Here, we reasoned that engineered allostery, a technique that has been applied to design light- and chemically-gated protein switches, could also be used to engineer phosphorylation-controlled protein switches (phospho-switches). Using an allosterically controllable Gal4 transcription factor as a scaffold, we show that a classic kinase FRET biosensor architecture can be used as a starting point for phospho-switch design. We optimize all features of the phospho-switch to develop an ERK-controlled transcription factor with a 20-fold phosphorylation-dependent change in transcriptional output. Our switch outperforms the c-fos promoter, a classic ERK-responsive transcriptional biosensor, in selectivity and sensitivity. We further show that our switch architecture can be generalized to other input kinases and allosterically controlled targets. This work provides a general platform for a new generation of kinase-responsive tools for biosensing and synthetic biology applications.