Directed evolution of estrogen receptor proteins with altered ligand-binding specificities
1Department of General and Developmental Physiology of Plants, Albrecht-von- Haller-Institute for Plant Sciences, Georg-August-University Goettingen, Untere Karspuele 2, 37073 Goettingen, Germany 2 Institute of Organic and Biomolecular Chemistry, Georg-August-University Goettingen, Tammannstr. 2, 37077 Goettingen, Germany 3Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S Mathews Avenue, Urbana, IL 61801, USA
4 To whom correspondence should be addressed. E-mail: cgatz{at}uni-goettingen.de or cgatz{at}gwdg.de
Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor 
(hER) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 µM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 µM) and a 10-fold decreased activity to 17ß-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific ‘gene switch’ as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.
Keywords: estrogen receptor/ligand specificity/saturation mutagenesis
Received July 2, 2008; revised September 18, 2008; accepted October 14, 2008.