Rational evolution for altering the ligand preference of estrogen receptor alpha

Area of Science:

• Synthetic Biology
• Molecular Biology
• Protein Engineering

Background:

• Estrogen receptor alpha is a key component in synthetic biology for controlling genome editing tools.
• Natural estrogen ligands disrupt cellular functions, limiting the receptor's synthetic biology applications.
• Modifying ligand preference is crucial for expanding the utility of estrogen receptors.

Purpose of the Study:

• To engineer an estrogen receptor variant with altered ligand specificity for enhanced synthetic biology applications.
• To develop a platform for modifying steroid receptor ligand preferences and profiling drug resistance.

Main Methods:

• Combined rational protein design, multi-site-directed mutagenesis, and directed evolution in Saccharomyces cerevisiae.
• Screened for variants with altered ligand responsiveness.
• Validated tamoxifen-activated variant (TERRA) in mammalian cells and mice.

Main Results:

• Developed TERRA, an estrogen receptor variant unresponsive to estrogens but activated by tamoxifen.
• TERRA's tamoxifen preference was confirmed in mammalian cells and in vivo models (mice).
• TERRA maintained functionality when fused to Cre recombinase, expanding its use in mammalian synthetic biology.

Conclusions:

• The developed platform enables engineering of ligand preference for steroid receptors, offering a versatile synthetic biology tool.
• TERRA provides a tamoxifen-inducible system for genome editing, overcoming limitations of estrogen-based systems.
• The methodology can be applied to profile drug-resistance landscapes in steroid receptor-targeted therapies.