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 co-localizing signaling molecules. To be effective, an inducible system should have dissociation constants in the "off" and "on" state that bracket the concentrations of the molecules that are being controlled. Here, we reengineer the light inducible dimer, iLID, to better control proteins present at high effective concentrations (5-100 μM). The new variant of the switch 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 co-localization of transmembrane proteins in neurons, where a higher affinity switch (0.8 μM to 47 ;M) was less effective because more co-localization was seen in the dark. Additionally, we lengthened the reversion half-life of the photoswitch. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution

“…Our solution exploits the recently uncovered dynamic exchange of various essential T3SS components between an injectisome-bound state and a freely diffusing cytosolic state 25,31 to control T3SS-dependent protein secretion by protein sequestration. SctQ, an essential and dynamic cytosolic component of the T3SS 31 , was genetically fused to one interaction domain of two optogenetic sequestration systems, the iLID and LOVTRAP systems 35,36,45 , while the membrane-bound interaction domain was co-expressed in trans . The two versions of the resulting LITESEC-T3SS system ( L ight- i nduced s ecretion of e ffectors through s equestration of e ndogenous c omponents of the T3SS ) can be applied in opposite directions: in the LITESEC-supp system, protein export is suppressed by blue light illumination; the LITESEC-act system allows to activate secretion by blue light.…”

“…They usually consist of homo-or hetero-dimers whose affinities are strongly altered upon irradiation by light of a certain wavelength. Mutations of specific amino acids in the optogenetic interaction domains can modulate binding affinities and the corresponding dissociation or return rates from a few seconds to several minutes 35,36 .…”

“…(ii) The iLID system, which employs the interaction of iLID, derived from a LOV2 domain from Avena sativa phototropin 1, with a smaller binding partner, SspB_Nano. The iLID system has a low binding affinity in the dark and a high affinity upon irradiation with blue light 34,36 . LOV and iLID systems therefore react to light in opposite directions, which allows to specifically release a bait protein (and, subsequently, to activate processes that require its presence) in the dark or upon illumination, respectively.…”

Engineering genetically-encoded tools for optogenetic control of protein activity

“…In practice, photodimerizers with larger differences between the dissociation constants in dark and light can be easier to optimize for photocontrol applications; however, appropriate expression level of these components is critical. For example, photodimerizers that interact with high affinity may be ideal for use in situations where proteins are expressed at low intracellular levels, but the same molecules may show unwanted interactions in dark and thus reduced dynamic range when expressed at higher levels [2]. …”

Construction of light-activated neurotrophin receptors using the improved Light-Induced Dimerizer (iLID)

“…To address these limitations, we have designed a strategy which uses the improved Light-Induced Dimer (iLID) and a tandem-dimer construct of SspB 3,11 to achieve location-specific activation of TrkA and TrkB in a light-dependent manner in live cells. We offer this tandem-dimer construct as a generalizable tool for activation of other RTKs with greater control of kinetics 12 , affinity, and localization than other available opto-RTK systems.…”