Protease-based synthetic sensing and signal amplification

Significance Synthetic biology envisages the creation of custom-based signaling by means of modular plug-and-play. This concept has primarily been realized in the construction of synthetic gene circuits. However, all real-time events in biology are processed by protein-based sensing and signal transducing systems; yet, the systematic bottom-up design of protein-based signaling systems remains elusive to date. Here we report a strategy for construction of modular protein switches based on artificially autoinhibited proteases whose activity can be modulated by specific proteolysis, ligand binding, or protein–protein interactions. We demonstrate that such protease-based ligand receptors or signal transducers can be assembled into different types of integrated signal sensing and amplification circuits that, in principle, can be connected to any biological process. The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor’s mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.

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