Small-Molecule Gated Artificial Regulatory Domains: A Novel Tool For Dissecting Signaling Pathways

Abstract

The activities of the proteins involved in eukaryotic signaling networks are controlled by a complex system of 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 (NS3-4A). 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 in two distinct approaches. Initial characterization entailed application of this system, in an inter-molecular fashion, to the regulation of AKT pathway activation in a localization dependent manner. A second mode of application was demonstrated using this switch in an intra-molecular system. The artificial regulator domains were applied 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 bio-orthogonal chemically inducible activator of Ras.