Azopyrazole-based Photochromic Dimerizer Enables Optical Control of Cellular Signaling with Minimal Basal Activation

Photochromic chemically induced dimerization (CID) system enables reversible optical control of protein–protein interactions in living cells, offering a powerful alternative to conventional optogenetic tools. We previously developed a photochromic CID system using an Escherischia coli dihydrofolate reductase (eDHFR)/HaloTag pair and an azobenzene-based photochromic dimerizer, pcDH, which achieved rapid and reversible protein translocation. However, undesirable background binding resulting from the high affinity of the E-isomer for eDHFR limited its precision. Here, we report an improved system employing an azopyrazole-based dimerizer, pcDH2b, and an engineered eDHFR mutant to reduce background binding. Molecular docking identified F31 as a key residue, prompting cell-based screening of F31 mutants. The eDHFR(F31I) showed minimal background binding while preserving efficient photoreversible dimerization. Fluorescence polarization assays confirmed reduced binding affinity in both dark and violet light-induced photostationary states. The optimized eDHFR(F31I)/Halo–pcDH2b pair exhibited rapid association and dissociation kinetics and enabled precise, lowbackground optical regulation of mitophagy in HeLa cells. Molecular dynamics simulations provided insights into the energetic and structural basis of E/Z selectivity. These results demonstrate that tuning ligand–protein affinity through protein mutation effectively minimizes background binding while maintaining fast photo-responses, providing a practical platform for reversible optical control of cellular signaling with minimal basal activation.