Design and evaluation of the Opto-MDMi (LOVTRAP) system.

A. Schematic of the p53-MDM2/MDMX negative feedback loop. B. Amino acid sequence and in vitro Kd value of PMI peptides for MDM2 and MDMX. C. Schematic illustration of the Opto-MDMi (LOVTRAP) system. D. Light-dependent nuclear accumulation of the Opto-MDMi (LOVTRAP) actuator. Blue boxes indicate periods of blue light illumination. H1-mCherry serves as a nuclear marker. Scale bar, 20 µm. E. Quantification of the actuator levels in the nucleus. Data are presented as mean ± s.d. (n = 10 cells). F. Representative images showing changes in actuator localization and p53 transcription reporter signal. Scale bar, 10 µm. G. Distribution of p53 reporter fluorescence intensity at 0 and 24 hours from the start of blue light illumination. Horizontal lines and crosses indicate the medians and means, respectively. Boxes represent the 25th and 75th percentiles; whiskers indicate the range between the maximum and minimum values excluding outliers. H. Temporal changes of the p53 transcription reporter in each cell line. Data represent mean ± s.e.m. (PMI-NLS, n = 122 cells; NLS, n = 125 cells).

In vitro screening of light-responsive inhibitory modules.

A. Schematic of the light-responsive LOV2-PMI chimera module. B. Workflow of the in vitro binding assay. T7 denotes the T7 tag used for detection. C. Representative CBB-stained SDS-PAGE gel of purified recombinant bait protein. D. Library of the LOV2 Jα-helix truncation mutants fused with PMI or PMI-M3 peptides. The critical residue Ile539 is highlighted in blue. E. Representative western blot detecting LOV2-PMI/PMI-M3 fragments bound to GST-MDM2(1–125) or GST-MDMX(1–125). The LOV2(–8Jα)-PMI/PMI-M3 fragment, truncated up to I539, serves as a control where light responsiveness is expected to be lost. GST is a negative control bait fragment. GFP-PMI/PMI-M3 and GFP are positive and negative control prey fragments, respectively. F. Differences in binding activity between dsm or lsm of each LOV2-PMI truncation mutant to GST-MDM2(1–125) (upper plot) or GST-MDMX(1–125) (lower plot). Blue and red points indicate experimental replicates.

Light-dependent interaction between LOV2-PMI fragments and MDM2 in cultured cells.

A. Schematic illustration of the live cell-based translocation assay. B. Light-dependent changes in localization of the Opto-MDMi (LOV2-PMI) actuator fragment identified through in vitro screening. Blue boxes indicate the time points at which light illumination was applied. Scale bar, 20 µm. C. Quantification of the Opto-MDMi (LOV2-PMI) actuator cytosolic level. Data are presented as mean ± s.d. (LOV2, n = 15 cells; LOV2(–6Jα)-PMI, n = 30 cells; LOV2(–7Jα)-PMI-M3, n = 20 cells).

Proposed mechanism of light-dependent interaction revealed by molecular dynamics simulations.

A. Protein structures of screened LOV2-PMI fragments predicted by AlphaFold 3. The amino acid residues drawn in light green and magenta indicate the LOV2 core domain and the PMI peptide, respectively. The Phe3, Trp7, and Leu10 residues in PMI/PMI-M3 peptides, known to be particularly important for interactions with MDM2/MDMX, were displayed in the ball-and-stick model to highlight their side chains. B. Snapshots of the Opto-MDMi (LOV2-PMI) protein structure obtained by molecular dynamics (MD) simulation. Protein structures were displayed every 150 nanoseconds based on the results of the MD simulation. C. Calculated root mean square fluctuation (RMSF) value of the Cα atom in the PMI/PMI-M3 peptides. D. Calculated solvent accessible surface area (SASA) value for the Phe3, Trp7, and Leu10 residues in PMI/PMI-M3 peptides.

Expanded screening of LOV2-PMI actuator using an inhibitory peptide library.

A. Table of inhibitory peptide variants with different in vitro affinities for MDM2/MDMX. SPR, surface plasmon resonance; ITC, isothermal titration calorimetry. B. Expression library of the LOV2-inhibitory peptide variants. Residues differing from the original PMI sequence are shown in red. C. Representative western blot images for the detection of each LOV2-inhibitory peptide fragments bound to GST–MDM2(1–125) or GST-MDMX(1–125). GFP-PMI and GFP serve as positive and negative control prey fragments, respectively. D. Comparison of binding activities between the dsm and lsm of each LOV2-inhibitory peptide fragment toward GST–MDM2(1–125) (upper) and GST-MDMX(1–125) (lower). Blue and red points represent experimental replicates. E. Light-dependent translocation of the Opto-MDMi (LOV2-PMI) actuator fragment obtained by the in vitro binding assay. Blue boxes indicate periods of light illumination. Scale bar, 20 µm. F. Quantification of the Opto-MDMi (LOV2-PMI) actuator levels in the cytoplasm. Plots show the mean ± s.d. (LOV2(–6Jα)-PMI(E5A), n = 40 cells; LOV2(–7Jα)-PMI-M5, n = 20 cells; LOV2(–7Jα)-PMI(E5A), n = 30 cells).

Light-dependent transcriptional activation of p53 by Opto-MDMi (LOV2-PMI)

A. Schematic illustration of the Opto-MDMi (LOV2-PMI) system expressed in cultured cells. B. Light-dependent changes in the actuator and p53 transcription reporter signals. The blue boxes indicate the time points at which light illumination was applied. Scale bar, 10 µm. C. Distribution of the fluorescence intensity of the p53 transcription reporter at 0 hours and 24 hours from the start of blue light illumination. Horizontal lines and crosses indicate the medians and means of the distribution, respectively. Horizontal lines and crosses indicate the medians and means, respectively. Boxes represent the 25th and 75th percentiles; whiskers indicate the range between the maximum and minimum values excluding outliers. D. Temporal changes in the p53 transcription reporter in each cell line. The plot shows the mean ± s.e.m. (LOV2(–6Jα)-PMI, n = 139 cells; LOV2(–7Jα)-PMI-M3, n = 116 cells; LOV2(–6Jα)-PMI(E5A), n = 170 cells; NLS, n = 82 cells).

Light-dependent transcriptional activation of p53 by combined Opto-MDMi system

A. Schematic illustration of the combination of Opto-MDMi (LOVTRAP) and Opto-MDMi (LOV2-PMI). B. Light-dependent changes in Opto-MDMi actuator localization and p53 transcription reporter signals. The blue boxes indicate the time points at which light illumination was applied. Scale bar, 10 μm. C. Distribution of the fluorescence intensity of the p53 transcription reporter at 0 hours and 24 hours from the start of blue light illumination. Horizontal lines and crosses indicate the medians and means of the distribution, respectively. Horizontal lines and crosses indicate the medians and means, respectively. Boxes represent the 25th and 75th percentiles; whiskers indicate the range between the maximum and minimum values excluding outliers. D. Temporal changes in the p53 transcription reporter in each cell line. The plot shows the mean ± s.e.m. (LOV2(–6Jα)-PMI, n = 181 cells; LOV2(–7Jα)-PMI-M3, n = 185 cells; LOV2(–6Jα)-PMI(E5A), n = 123 cells; NLS, n=209 cells). E. Quantification of cell division events during the period from 0 to 24 hours in panel D.