Modulating the Sensitivity of a Bacterial Biosensor for Peroxisome Proliferator-Activated Receptors Gamma to the Ligands by Engineering Spacer Linker

Peroxisome proliferator-activated receptors gamma (PPARG), belonging to the human nuclear receptor superfamily, is a vital ligand-modulated transcription activator that regulates adipogenesis and lipid homeostasis, as well as other physiological functions. The development of a biosensor to detect PPARG ligands will greatly facilitate drug discovery for diabetes, inflammation, hyperlipidemia and other disorders. In this work, we have constructed a bacterial PPARG ligand biosensor based on an allosteric fusion protein that we have previously reported. This fusion protein allows ligand binding to a specific target to be reflected in growth phenotypes of auxotrophic E. coli strains. Although our previously designed biosensors worked well, the PPARG sensor did not behave consistently, and was unable to correctly identify a number of important ligands. Moreover, pronounced constitutive growth in the absence of the ligands was observed. We have hypothesized that this is due to specific structural features that are unique to the PPARG receptor LBD. To improve the sensitivity of PPARG sensor to the ligands, the spacer linkers between the intein and PPARG LBD were subjected to optimization. By inserting Gly-Ser linkers of various lengths, and adjusting the incubation temperatures as needed, we were able to generate a biosensor strain with growth phenotypes that are strongly dependent on PPARG ligand binding. The half-maximal effective concentrations (EC50) for several typical ligands were comparable to published results for direct binding and transactivation assays. It was concluded that the C-terminal spacer of the PPARG LBD plays a critical role in signal attainment, and the flexible GS linkers help to refine the arrangement of helix 12 to reach an active conformation upon ligand binding. This optimized biosensor protein is practical for the screening of drug-like compounds that may target and modulate the PPARG receptor.