Journal of Biological Chemistry
Volume 279, Issue 32, 6 August 2004, Pages 33855-33864
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Protein Structure and Folding
Directed Evolution of Human Estrogen Receptor Variants with Significantly Enhanced Androgen Specificity and Affinity*

https://doi.org/10.1074/jbc.M402118200Get rights and content
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Human estrogen receptor α (hERα) 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 hERα 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 hERα variants with increased transactivation potency toward testosterone. After two rounds of directed evolution, we identified five hERα 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 17β-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 hERα 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.

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*

This work was supported by National Science Foundation CAREER Award BES-0348107 (to H. Z.), the Department of Chemical and Biomolecular Engineering of University of Illinois (to H. Z.), and National Institute of Health Grants CA18119 (to B. S. K.) and DK15556 (to J. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 7 and 8.