A Functionally Orthogonal Estrogen Receptor-Based Transcription Switch Specifically Induced by a Nonsteroid Synthetic Ligand

Abstract

It is highly desirable to design ligand-dependent transcription regulation systems based on transactivators unresponsive to endogenous ligands but induced by synthetic small molecules unable to activate endogenous receptors. Using molecular modeling and yeast selection, we identified an estrogen receptor ligand binding domain double mutant (L384M, M421G) with decreased affinity to estradiol and enhanced binding to compounds inactive on estrogen receptors. Nonresponsiveness to estrogen was achieved by additionally adding the G521R substitution while introducing an "antagonistic-type" side chain in the compound, as in 4-hydroxytamoxifen. The triple-substituted ligand binding domain is insensitive to physiological concentrations of estradiol and has nanomolar affinity for the ligand. In this binary system, both receptor and ligand are, therefore, reciprocally specific. The mutated variant in the context of a chimeric transcription factor provides tight, ligand-dependent regulation of reporter gene expression.

Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

“…The representative photobleaching profiles were shown in the right panels. As a control, the distribution of observed bleaching steps of HBD*-GFP was shifted from one-step (∼80%) to two-step (∼60%) upon addition of 4-OHT (Figure 3A, top panels), which is consistent with the fact that 4-OHT induces HBD* dimer formation as reported previously 32 .…”

“…A G521R mutant of HBD (termed HBD*) that binds to a synthetic antiestrogen 4-hydroxytamoxifen (4-OHT) but not oestrogen has been used to avoid the effect of serum oestrogen in culture medium 32 . We fused HBD* to the C-terminus of MLKL and expressed it in MLKL KO L929 cells (Figure 2A and Supplementary information, Figure S2A).…”

“…4-OHT-induced dimer formation is well-established in in vitro system 31,32 ; however, the subunit number of the induced complex could be more than two if the HBD*-fused proteins are able to interact with other protein(s) through domains other than HBD*. To examine the stoichiometry of the ND complex induced by 4-OHT in our experiments, we used the SiMPull assay that combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy 33 .…”

Directed Evolution of Orthogonal Ligand Specificity in a Single Scaffold

“…Specificity‐reengineering approaches involving NHRs have typically involved mutation of the LBD to a form that is not activated by the natural ligand but instead by a synthetic small molecule that is inactive against the wild‐type LBD. The LBD of the human estrogen receptor α (hERα) has proven to be a particularly versatile platform for the creation of orthogonal ligand–receptor pairs 12. 13 Despite these efforts and studies on other NHR LBDs, the creation of unique, NHR‐based independently functioning ligand–receptor pairs that are not only orthogonal to cellular elements but that do not cross‐interact with one another has yet to be demonstrated.…”

Destabilizing Domains Derived from the Human Estrogen Receptor

“…11 Furthermore, previous studies disclosed ligands that bind to mutant but not wild-type forms of the ERLBD. 12 By using one of these mutant domains encoding three mutations (L384M, M421G, G521R) 12 , we anticipated that it would be possible to regulate the stability of an ERLBD-derived DD using a ligand that does not perturb endogenous estrogen-sensitive networks. An additional mutation (Y537S) was introduced to further destabilize the ERLBD and to configure it as a potential DD candidate.…”