Design and Development of Chemically Gated Artificial Regulatory Domains

The activities of many proteins involved in eukaryotic signaling networks are controlled by allosteric regulatory domains, in which auto‐inhibitory interacting domains repress protein activity. The field of synthetic biology aims to harness such biological systems to study the roles of individual signaling nodes, and more generally, to obtain spatio‐temporal control over protein activity. To this end, we have developed a novel, modular chemical genetic method for conferring small‐molecule control over the activity of signaling proteins. This system replaces their auto‐inhibitory regulatory domains with an artificial protein‐protein interaction engineered from the Hepatitis C Virus protease (NS3). This artificial regulatory mechanism can be disrupted with a bio‐orthogonal, cell‐permeable, and clinically approved small molecule. This system confers spatio‐temporal control over protein activity in a dose‐dependent and reversible manner. Initial engineering efforts were conducted by conferring our switch to the guanine nucleotide exchange factor Son of Sevenless, an activator of the GTPase Ras. A computational method using Rosetta Remodel directed the replacement of the endogenous regulatory modules with the HCV‐Protease based switch, resulting in the successful generation of a chemically inducible activator of Ras. To illustrate the utility and versatility of this synthetic switch, we have applied our system to a series of structurally and functionally distinct targets, including the DNA Endonuclease Cas9.A chemically inducible artificial regulatory domain system has been engineered from an HCV‐protease/peptide interaction. This novel, bio‐orthogonal switch was initially characterized using the GEF SOS. Expression of the construct results in an inactive GEF. Addition of a bio‐orthogonal small‐molecule drug disrupts the intra‐molecular binding event, thus activating the GEF.