title: Directed evolution of estrogen receptor proteins with altered ligand-binding specificities.
abstract: 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 alpha (hER alpha) 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 alpha 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 microM. 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 microM) and a 10-fold decreased activity to 17beta-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.
authors.affiliation: Department of General and Developmental Physiology of Plants, Albrecht-von- Haller-Institute for Plant Sciences, Georg-August-University Goettingen, Untere Karspuele 2, 37073 Goettingen, Germany.
authors.forename: Kazi Mohammed Didarul
authors.initials: KM
authors.lastname: Islam
authors.forename: Meik
authors.initials: M
authors.lastname: Dilcher
authors.forename: Corinna
authors.initials: C
authors.lastname: Thurow
authors.forename: Carsten
authors.initials: C
authors.lastname: Vock
authors.forename: Ilga Kristine
authors.initials: IK
authors.lastname: Krimmelbein
authors.forename: Lutz Friedjan
authors.initials: LF
authors.lastname: Tietze
authors.forename: Victor
authors.initials: V
authors.lastname: Gonzalez
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
authors.forename: Christiane
authors.initials: C
authors.lastname: Gatz
mesh_terms: D019020:Directed Molecular Evolution; D004958:Estradiol / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051548:Histone Acetyltransferases / Q000378:metabolism; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D056920:Nuclear Receptor Coactivator 1; D015202:Protein Engineering; D000077185:Resveratrol; D013267:Stilbenes / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D008024:Ligands; D013267:Stilbenes; D014157:Transcription Factors; D004958:Estradiol; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D000077185:Resveratrol