title: Directed evolution of human estrogen receptor variants with significantly enhanced androgen specificity and affinity.
abstract: Human estrogen receptor alpha (hERalpha) and human androgen receptor exhibit exquisite ligand specificity, which underlies their remarkable ability to effect ligand-regulated gene transcription in a highly distinctive and specific manner. Here we used a directed evolution approach to create hERalpha variants with enhanced androgen specificity and affinity with the goal to better understand the molecular basis of ER ligand specificity and the evolutionary mechanism of nuclear receptors. We developed a sensitive yeast two-hybrid system to screen for hERalpha variants with increased transactivation potency toward testosterone. After two rounds of directed evolution, we identified five hERalpha variants with dramatically improved transactivation potency toward testosterone in both yeast and mammalian cells. These variants showed up to 7,600-fold improvement in the binding affinity for testosterone and only slightly reduced affinity toward 17beta-estradiol. Detailed analysis of these evolved variants and a few site-directed mutants generated de novo led to several unexpected findings including the following. 1) Only two beneficial mutations were needed to create hERalpha variants with near nanomolar affinity for testosterone. 2) Some beneficial mutations were synergistic, context-dependent, or non-additive. 3) Of the five identified beneficial mutations, four of them were not in the ER ligand binding pocket and yet exerted important action on ligand specificity. 4) The single ligand-contacting mutation E353Q plays a dominant role in discriminating androgens and estrogens. These results, viewed in conjunction with the ligand exploitation model of nuclear receptor evolution, suggest that the mutation E353Q may represent a key event in the evolution of androgen receptors from an ancestral estrogen receptor and that ligand promiscuity may play an important role in the creation of new nuclear receptors via divergent evolution.
authors.affiliation: Center for Biophysics and Computational Biology and the Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801, USA.
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Chen
authors.forename: Benita S
authors.initials: BS
authors.lastname: Katzenellenbogen
authors.forename: John A
authors.initials: JA
authors.lastname: Katzenellenbogen
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism*; D001665:Binding Sites; D003460:Crystallization; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D014644:Genetic Variation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011944:Receptors, Androgen / Q000235:genetics; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013739:Testosterone / Q000378:metabolism / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: D000728:Androgens; D047628:Estrogen Receptor alpha; D011944:Receptors, Androgen; D011960:Receptors, Estrogen; D013739:Testosterone; D004958:Estradiol