Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches

Abstract

To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre-defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.

Keywords: chemical inducers of dimerization; induced dimerizer; optogenetic; protein control.

Figure 1.

Controlling protein activity with chemical or light-induced association and dissociation. (A) Chemically-induced dimerization system. Addition of a small molecule (CID, chemical inducer of dimerization) that bridges two dimerization domains induces their association. (B) Light induced dimerizer system (photodimerizer system). With illumination, a photosensitive domain transitions to an excited state with altered confirmation, allowing high affinity interaction with a partner. The reaction is inducibly reversed in dark, as the protein recovers the low-affinity, dark state conformation. In some cases the binding affinities can be reversed, such that the partner binds with higher affinity to the dark state conformation. (C) Utilization of a induced dimerization system to reconstitute a split protein, resulting in conditional control of protein activity.

Figure 2.

Additional regulation layered upon inducible dimerization systems. Inducible dual system approaches are divided into two categories, regulation of system component properties (protein localization, activity, or binding) without altering actual protein levels and (A-E) and regulation of actual protein abundance (F-I). (A) Release from organelle compartmentalization. In the example shown, a dimerizer component is prevented from entering its functional organelle (the nucleus) through anchoring to a cytosolic tether. The protein can be inducibly released through several mechanisms, including use of light or ligand-activated dimerization systems and inducible protease cleavage. (B) Induced change in subcellular localization. This concept is similar to (A), but does not involve compartmentalization within an organelle. (C) Release of steric block. In the example shown, a light- or ligand-sensitive protein acts to prevent association of a dimerizer-dependent split protein in the uninduced state. With the inducer, the steric block is removed and the protein can associate. (D) Ligand-induced allosteric change. (E) Induced oligomerization. (F) Inducible transcriptional regulation. A light- or ligand-induced transcriptional system is layered onto a dimerization system. (G) Ligand-induced protein stabilization. A destabilized ligand-binding domain is added to one or more components, resulting in protein degradation in the absence of inducer. (H) Protease-induced protein stabilization. Similar to (G), a degron is attached to a component, resulting in constitutive degradation unless a protease is active, removing the degron. The cleavage can be regulated by a dimerized split protease, a photosensitive cleavage site (e.g., PhoCl or AsLOV-caged cleavage site), or a protease inhibitor. (I) Inducible protein degradation. A protein is fused to a domain that can be conditionally targeted to the proteasome (with addition of an inducer).