Fig. 1: Design and optimization of GAL4-derived PRCIS.
a Schematic illustration of the rational design and optimization of GAL4-derived PRCIS. The dimerization region of GAL4 is first truncated and extended with E5/K5. For single constraint, conformational constraining of DBDs is achieved by fusing one DBD to E5 with a PCP knot (eGALo/+), or tethering the other DBD with a PCP knot to K5 via the assembly of GFP β1-10 and GFP β11 (eGAL+/o). Then, the other free DBD is constrained with a protease uncleavable peptide, generated eGAL–/+ and eGAL+/–. Finally, a dual PCP-locked variant is engineered (eGAL+/+). Upon protease-induced PCP cleavage, eGAL+/+ will restore the DNA binding and activate reporter transcription. b Crystal structure of GAL4 (1-99) binding with DNA (PDB entry: 3COQ). Orange arrows indicate the truncated positions between residues 50 and 51, 65 and 66, and 70 and 71. c Constructs of conformationally constrained eGAL designs. For eGAL+/o, the two protein fragments are expressed separately using an T2A sequence from a single plasmid, which are then intermolecularly assembled into the dimerized ATF. d Evaluating the TEVp-responsive performance of different conformationally inhibited eGAL designs. n = 3, individual data points represent independent triplicates performed on the same day. Error bars represent mean mCherry fluorescence intensity (MMFI) in arbitrary units (arbs.units) ± standard deviation (SD). Significance (two-tailed, unpaired t test) and TEVp-induced fold changes of mCherry fluorescence are labeled for each comparison, **p < 0.01, ***p < 0.001, ****p < 0.0001. a, c Created in BioRender. He, J. (2025) https://BioRender.com/t0g6x4h. Source data are provided as a Source Data file.
