Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization

Abstract

Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.

Figure 1

In vitro characterization of iLID SspB_milli and sLID. A) Left panel: Binding of iLID to SspB SspB_milli under blue light (blue) and in dark (black) measured by fluorescence polarization. Right panel: Binding of sLID to SspB_nano (circles) or SspB_milli (squares) under blue light (blue) and in dark (black). B) Reversion of FMN-LOV2 covalent adduct is measured by recovery of absorbance at 450nm after 30 sec of blue light activation for WT iLID (blue) and sLID (green). C) Schematic displaying dynamic range of each characterized photoswitch. Blue circles denote the lit state affinity, dark circles mark the dark state affinity, and the connecting line represents change due to blue light.

Figure 2

Membrane localization A) Representative images of membrane recruitment and reversion analyzed in B and C. Cells transfected with each membrane bound switch pair were imaged and activated by confocal microscopy. Blue square marks the ROI activated with blue light. B) Ratio of RFP fluorescence intensity inside the activated ROI to outside the ROI during activation. C) Ratio of RFP fluorescence intensity inside the activated ROI to outside the ROI after activation. D and E) Venus-iLID-CAAX/RFP-SspB_micro or Venus-sLID-CAAX/SspB_milli were activated with 15, 30, or 60 s between activations. Plots represent the normalized ratio of RFP fluorescence intensity inside the activated ROI to outside the ROI during activation. F) The maximum normalized fluorescence intensity ratio from D and E determined by fitting the curves. (Data for Nano and Micro has been previously published .)

Figure 3

Mitochondrial localization A) Representative images of mitochondrial recruitment and reversion analyzed in B. Cells transfected with each mitochondrial bound switch pair were imaged and activated by confocal microscopy. B) Ratio of mitochondrial to cytoplasmic RFP fluorescence intensity for each switch pair. Fold change in localization is represented by the bars to the right. (Data for Nano and Micro has been previously published .)

Figure 4

Induced localization of membrane protein, GluA1, is enhanced by use of sLID/SspB_milli. A) Representative images of GluA1-mCh-SspB localization using SspB nano (bottom), micro (middle) and milli (top) as analyzed in B, C and D. Arrowheads mark PSD. B) Representative change in GluA1-mCh-SspB fluorescence intensity over the spine and shaft before and after activation for SspB nano (bottom), micro (middle) and milli (top). Linescan profiles were generated using the grey dotted lines in A. C) PSD-localized mCherry fluorescence intensity is plotted over time normalized to the initial intensity for GluA1-mCh fused to SspB nano (n=89 spines from 5 neurons), micro (n=50 spines from 5 neurons) and milli (n=53 spines from 7 neurons). D) Ratio of spine to shaft mCherry fluorescence intensity before and after activation for GluA1-mCh fused to SspB nano (n=30 spines from 3 neurons), micro (n=30 spines from 2 neurons) and milli (n=30 spines from 5 neurons). ***p<0.001, paired student’s t-test.