Chemical and optogenetic methods for post-translationally controlling protein function have enabled new discoveries and the engineering of synthetic cellular functions. However, most of these methods only confer single-input, single-output control. To increase the diversity of posttranslational behaviors that can be programmed we built a system based on a single protein receiver that can integrate multiple drug inputs, including approved therapeutics. Our system translates drug inputs into diverse outputs with engineered reader proteins that provide variable dimerization states of the receiver protein. We show that our single receiver protein architecture can be used to program diverse cellular responses, including graded and proportional dual-output control of transcription and mammalian cell signaling. We apply our tools to titrate the competing activities of the Rac and Rho GTPases to control cell morphology. Our receiver protein and suite Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

No abstract

Chemical methods that allow the spatial proximity of proteins to be temporally modulated are powerful tools for studying biology and engineering synthetic cellular behaviors. Here, we describe a new chemically-controlled method for rapidly disrupting the interaction between two basally colocalized protein binding partners. Our chemically-disrupted proximity (CDP) system is based on the interaction between the hepatitis C virus protease (HCVp) NS3a and a geneticallyencoded peptide inhibitor. Using clinically-approved antiviral inhibitors as chemical disrupters of the NS3a/peptide interaction, we demonstrate that our CDP system can be used to confer temporal control over diverse intracellular processes. This NS3a-based CDP system represents a new modality for engineering chemical control over intracellular protein function that is complementary to currently available techniques.

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here, we report a computationally-guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop Chemically Inducible Activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS/ERK signaling dynamics compared to growth factor stimulation, and that these dynamics differ between cell types. We also found that the clinically-approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach to design intramolecularly-regulated protein tools by applying this methodology to the Rho Family GEFs.