(E-189) Engineering of a bacterial two component system for optogenetic control of biological processes

Introduction:: Genetically-encoded calcium actuators and modulators (GECAs) have enabled precise modulation of calcium signaling with high temporal and spatial resolution. Here, we report the optimization and integration of a dimerization system based on an engineered bacterial ssrA-sspB interaction pair for optogenetic gating of ion channel, as well as other biological processes in mammalian cells. Given the small size of ssrA as a 10-mer peptide, it can be conveniently inserted into proteins of interest without perturbing host protein function to enable optogenetic control of protein activity when combined with photo-switchable sspB.


Materials and Methods:: We performed a systemic mutagenesis screening of the ssrA-sspB pair by bioluminescence resonance energy transfer (BRET) in order to identify ssrA mutants with varying binding strength for sspB. We further fused the ssrA tag on mitochondria and NanoLuc with sspB to measure the BRET signal. We optimized sspB by fusing it with Arabidopsis cryptochrome 2 (CRY2) or the oat light-oxygen-voltage-sensing domain 2 (LOV2) to generate both optogenetic ON and OFF switches to control cell signaling.


Results, Conclusions, and Discussions:: Results:

We used optimized ssrA-sspB dimerization system to develop three sets of tools by taking advantage of light-inducible protein-protein association or dissociation. First, the ssrA tag was inserted into the stromal interaction molecule 1 (STIM1) to enable light inducible activation of ORAI calcium channel in the plasma membrane, as well as the downstream effector activation. Second, we generated a handful of ssrA-MLKL fusion protein to induce cell death by forming a pore on the plasma membrane under blue light stimulation. Additionally, two potential applications are being studied. First, we incorporated the ssrA/sspB system into the bacterial MinD/MinE oscillatory system to control the frequency of MinD/E oscillation with a simple pulse of light. Second, the system can be potentially applied to engineered G protein-coupled receptors (GPCR) to enable remote control of GPCR signaling with light stimulation.



Conclusions and Discussions:

The optimized ssrA-sspB (designated AB Tag) dimerization system provides a versatile tool to enable precise modulation of calcium influx and other biological processes with high precision, rapid reversibility, and non-invasiveness. In addition, this optogenetic system has a wide range of applications, including antigen recognition, cell death induction, and altering the frequency of MinD/E protein oscillator. Our findings provide a new approach for the design and optimization of molecular tools with improved precision and versatility for controlling cell signaling.


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