Protease-based synthetic sensing and signal amplification

Viktor Stein, Kirill Alexandrov

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.