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• W2045128521 abstract "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. 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. The estrogen receptor (ER) 1The abbreviations used are: ER, estrogen receptor; AR, androgen receptor; DHT, 5α-dihydrotestosterone; ERE, estrogen response element; EC50, half-maximal effect concentration; E2, 17β-estradiol; HEC-1, human endometrial cancer; LBD, ligand binding domain; MOE, molecular operating environment; RBA, relative binding affinity; SRC-1, steroid receptor coactivator 1; T, testosterone; Pg, Progesterone; h, human. 1The abbreviations used are: ER, estrogen receptor; AR, androgen receptor; DHT, 5α-dihydrotestosterone; ERE, estrogen response element; EC50, half-maximal effect concentration; E2, 17β-estradiol; HEC-1, human endometrial cancer; LBD, ligand binding domain; MOE, molecular operating environment; RBA, relative binding affinity; SRC-1, steroid receptor coactivator 1; T, testosterone; Pg, Progesterone; h, human. and androgen receptor (AR) belong to the steroid hormone nuclear receptor superfamily that regulates hormone-responsive genes in a ligand-inducible manner (1Katzenellenbogen J.A. Katzenellenbogen B.S. Chem. Biol. 1996; 3: 529-536Abstract Full Text PDF PubMed Scopus (152) Google Scholar). Like other members of this nuclear receptor super-family, the ER and AR have three modular structural domains: an amino-terminal ligand-independent transactivation domain, a central DNA binding domain, and a carboxy-terminal ligand binding domain (LBD). The LBD of either ER or AR interacts specifically with its physiological ligand and contains a dimerization function and a ligand-dependent activation function AF-2 (2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6028) Google Scholar, 3Tenbaum S. Baniahmad A. Int. J. Biochem. Cell Biol. 1997; 29: 1325-1341Crossref PubMed Scopus (86) Google Scholar, 4Moras D. Gronemeyer H. Curr. Opin. Cell Biol. 1998; 10: 384-391Crossref PubMed Scopus (698) Google Scholar, 5Aranda A. Pascual A. Physiol. Rev. 2001; 81: 1269-1304Crossref PubMed Scopus (1162) Google Scholar). Interestingly, despite their low sequence homology (<20%), the ER LBD and the AR LBD share a similar structural motif consisting of 12 α-helices arranged in an antiparallel sandwich motif (Figs. 1A and 6A).Fig. 6A, ribbon diagrams of the superimposed three-dimensional structures of hAR LBD (yellow) complexed with testosterone (cyan) and hERα LBD (red) complexed with E2 (pink). The beneficial mutations except for A569T are shown in Corey-Pauling-Koltun model (green). A569T is not in the LBD domain. B, diagrams showing the interactions between the ligand (testosterone or E2) with the residues within the binding cavity (van der Waals cut-off distance, 4.0 Å) and the receptor (hAR LBD or hERα LBD). Residues Met-343, Leu-346, Trp-383, Phe-404, Met-421, Leu-428, and Met-528 from hERα LBD and Leu-701, Trp-741, Phe-764, Met-780, and Met-787 from hAR LBD are omitted from the graph for clarity. Both diagrams were made using Visual Molecular Dynamics (30Humphrey W. Dalke A. Schulten K. J. Mol. Graph. 1996; 14: 33-38Crossref PubMed Scopus (36896) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Both the ER and the AR are of particular interest because of their important roles in the growth, differentiation, metabolism, reproduction, and morphogenesis of higher organisms and humans and their association with several human diseases. Estrogens such as 17β-estradiol (E2) (Fig. 1B), acting through the ERα or ERβ, regulate the differentiation and maintenance of neural, skeletal, reproductive, and cardiovascular tissues. The regulation of ERα activity plays a critical role in osteoporosis, cardiovascular disease, and breast cancer (3Tenbaum S. Baniahmad A. Int. J. Biochem. Cell Biol. 1997; 29: 1325-1341Crossref PubMed Scopus (86) Google Scholar, 6Nilsson S. Makela S. Treuter E. Tujague M. Thomsen J. Andersson G. Enmark E. Pettersson K. Warner M. Gustafsson J.A. Physiol. Rev. 2001; 81: 1535-1565Crossref PubMed Scopus (1573) Google Scholar). Androgens and their receptors exert crucial actions in male physiology and pathology. The binding of male sex steroids 5α-dihydrotestosterone (DHT) and testosterone (T) (Fig. 1B) to the AR initiates male sexual differentiation and development (7Mcphaul M.J. Marcelli M. Tilley W.D. Griffin J.E. Wilson J.D. FASEB J. 1991; 5: 2910-2915Crossref PubMed Scopus (79) Google Scholar). Mutations in the human AR LBD have been linked to several diseases such as prostate cancer and the androgen insensitivity syndrome (3Tenbaum S. Baniahmad A. Int. J. Biochem. Cell Biol. 1997; 29: 1325-1341Crossref PubMed Scopus (86) Google Scholar). Thus, considerable effort has been directed at elucidating the molecular basis of ligand specificity of the ER and AR LBDs.The ligand specificity and sensitivity of the ER or AR toward its physiological ligand are extremely high. Both the AR and the ER bind their physiological ligands with subnanomolar affinity, and a small difference in the ligand structure can have a dramatic effect on ligand binding affinity. For example, although the chemical structures of testosterone and E2 differ only slightly in the A-ring region (Fig. 1B), the affinity of human ERα for testosterone is >10,000-fold weaker than that for E2 (this work), whereas the affinity of human AR for E2 is 44-fold weaker than that for testosterone (8Toth I. Faredin I. Mesko E. Wolfling J. Schneider G. Pharmacol. Res. 1995; 32: 217-221Crossref PubMed Scopus (5) Google Scholar). Comparison of the crystallographic structures of the human ERα LBD (hERα LBD) complexed with E2 and the human AR LBD (hAR LBD) complexed with testosterone indicates that the majority of the residues (14 of 20) interacting with the ligand are different between the ER and the AR (Fig. 1A). Thus, we have been intrigued by the structural features of hERα and hAR that underlie their ability to discriminate E2 from testosterone. Prior site-directed mutagenesis studies have indicated that the hERα residue Glu-353 plays a significant role in discriminating estrogens from androgens (9Ekena K. Katzenellenbogen J.A. Katzenellenbogen B.S. J. Biol. Chem. 1998; 273: 693-699Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). However, that single mutation alone cannot account for the observed difference in ligand affinity and specificity between the ER and AR. To gain a further understanding of the interactions between the receptor and ligand, we have taken a new approach, directed evolution.Directed evolution is a powerful tool for engineering proteins with improved functions, such as solubility, stability, affinity, and activity (10Arnold F.H. Acc. Chem. Res. 1998; 31: 125-131Crossref Scopus (449) Google Scholar, 11Schmidt-Dannert C. Biochemistry. 2001; 40: 13125-13136Crossref PubMed Scopus (66) Google Scholar). Here we report the application of directed evolution to engineer hERα variants with up to 7,600-fold improvement in the binding affinity toward testosterone. Strikingly, these evolved hERα variants exhibited only slightly reduced binding affinity (1.5–3-fold) to E2 and contained only two or three amino acid substitutions in the LBD. Further biochemical and structural analysis of these evolved ER variants provided new insights into the molecular basis of ER ligand specificity and the evolutionary mechanism of steroid receptors.EXPERIMENTAL PROCEDURESReagents—Cell culture media were purchased from Invitrogen. Calf serum was obtained from HyClone Laboratories, Inc. (Logan, UT), and fetal calf serum was purchased from Atlanta Biologicals (Atlanta, GA). The luciferase assay system was from Promega Corp. (Madison, WI). The 17β-estradiol, testosterone, dihydrotestosterone, and isopropyl-β-d-thiogalactopyranoside were from Sigma. All restriction enzymes and DNA-modifying enzymes were obtained from New England BioLabs (Beverly, MA). Yeast strain YRG-2 (Mat a ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3 112 gal4-542 gal80-538 LYS2::UASGAL1-TATAGAL1-HIS3 URA3::UASGAL4 17mers(x3)-TATACYC1-lacZ) were from Stratagene (La Jolla, CA). Taq DNA polymerase was from Promega Corp. [3H]E2 (50 Ci/mmol) was obtained from Amersham Biosciences. QIAprep spin plasmid miniprep kit, QIAEX II gel purification kit, and QIAquick PCR purification kit were purchased from Qiagen (Valencia, CA). Various oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, IA).Plasmid Construction—pCMV5-ERα plasmid containing the wild type full-length hERα cDNA (12Wrenn C.K. Katzenellenbogen B.S. J. Biol. Chem. 1993; 268: 24089-24098Abstract Full Text PDF PubMed Google Scholar) was first digested with EagI and BamHI and then treated with T4 DNA polymerase to form blunt ends at both termini followed by ligation into the EcoRI site (also treated with T4 DNA polymerase) of pBD-GAL4-Cam vector (Stratagene) to form pBD-GAL4 hERα containing hERα amino acids 312–595. pGAD424 SRC-1 containing the full-length SRC-1 was constructed as described elsewhere (13Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar). Mammalian cell reporter plasmid (ERE)2-pS2-Luc encoding a luciferase gene downstream of 2ERE was described in Ref. 14Sun J. Huang Y.R. Harrington W.R. Sheng S. Katzenellenbogen J.A. Katzenellenbogen B.S. Endocrinology. 2002; 143: 941-947Crossref PubMed Scopus (181) Google Scholar, and the internal control plasmid pCMVβ was from Clontech.The genes encoding the evolved hERα LBD variants were subcloned into the pCMV5-ERα plasmids to reconstitute the full-length hERα genes. Briefly the LBD genes were PCR-amplified with primers ERαLBDCam5F (5′-ggc cga cca gat ggt cag tg-3′) and ERα3_ClaI (5′-gga tcg att cag act gtg gca g-3′, the ClaI restriction site is underlined), and the PCR products were purified using a QIAquick PCR purification kit. The pCMV5-ERα plasmid was digested with HindIII, and the 1-kb fragment was purified from a 1% agarose gel. The two purified DNA fragments share an overlap region of approximately 100 bp. Overlap extension PCR was performed to obtain full-length mutant hERα genes (10 cycles of 94 °C for 1 min and 72 °C for 4 min) followed by PCR amplification using primer ERα_pCMV5_5KpnI (5′-ccg gta ccc cat gac cat gac-3′, the KpnI restriction site is underlined) and ERα3_ClaI. The PCR products were then purified, digested with KpnI and ClaI, and ligated into the KpnI-ClaI-digested pCMV5-ERα vector.The LBDs of the wild type hERα and the evolved hERα variants were also subcloned into pET15b (Novagen) for protein expression in Escherichia coli. Briefly, for each plasmid construct, the LBD gene was amplified from pBD-GAL4 hERα (wild type or mutants) with primers ERα_NdeI_N (5′-act gta tcg ccg cat atg gcc gac cag atg-3′, the NdeI site is underlined) and ERα_BamHI_C (5′-taa gcg gga tcc tca gac tgt ggc agg gaa-3′, the BamHI site is underlined), purified, digested with NdeI and BamHI, and ligated into the NdeI-BamHI-digested pET15b vector.Library Creation and Screening—Error-prone PCR was used to generate a library of mutagenized LBD fragments consisting of hERα amino acids 312–595 using pBD-GAL4 hERα as a template and ERαLBDCam5F and ERαLBDCam3R (5′-tca gac tgt ggc agg gaa acc-3′) as primers according to the protocol described elsewhere (15Zhao H. Moore J.C. Volkov A.A. Arnold F.H. Demain A.L. Davies J.E. Manual of Industrial Microbiology and Biotechnology. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1999: 597-604Google Scholar). The resulting mutagenized LBD fragments were then co-transformed with the pBD-GAL4 hERα vector that was digested with BglII and BsaI to form a 94-bp gap into yeast YRG2 cells pretransformed with the pGAD424 SRC-1 plasmid. Yeast transformation was carried out using the lithium acetate/single-stranded carrier DNA/polyethylene glycol protocol (16Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2039) Google Scholar).For library screening, a two-tiered strategy consisting of an agar plate-based selection followed by a 96-well plate-based screening was used. In the selection method, the mutagenized LBD fragments were co-transformed with the BglII-BsaI-digested pBD-GAL4 hERα vector into Saccharomyces cerevisiae YRG2 cells harboring pGAD424 SRC-1. The transformed cells were plated on an agar plate containing minimum medium lacking tryptophan, leucine, and histidine and supplemented with an appropriate testosterone concentration. The testoster-one concentration was chosen such that yeast cells bearing the parental hERα LBD in each round of directed evolution cannot form colonies, whereas yeast cells bearing a variant with 5–10-fold improvement in ligand binding affinity may form colonies. Colonies that formed on the selection plates after incubation at 30 °C for 2 days were picked and streaked onto two agar plates containing the same minimum medium as mentioned above, one with and one without testosterone. The colonies appearing on the agar plate with testosterone but not on the agar plate without testosterone were picked and assayed in the 96-well plate to determine their EC50 values. In the 96-well plate assay, yeast YRG2 cells were grown to log phase (A600 = 2–5) in synthetic complete minimal medium lacking leucine and tryptophan at 30 °C overnight (12–16 h) with shaking. The resulting cell culture was diluted to A600 = 0.002 in liquid minimal medium lacking leucine, tryptophan, and histidine. Each well in the 96-well plate contained 200 μl of diluted yeast cells and 0.2 μl of the appropriate ligand at a specific concentration dissolved in pure dimethylformamide or EtOH. 96-well plates were incubated at 30 °C for 20–24 h, and the cell density at 600 nm was measured using a SpectraMax 340 PC plate reader (Amersham Biosciences).Site-directed Mutagenesis—The single, double, and triple mutants were created using overlap extension PCR and yeast in vivo recombination. Briefly, for each mutation, a pair of mutagenic primers with each containing the desired mutation was prepared and used in combination with two oligonucleotide primers flanking the LBD gene. Each of these two flanking primers contained 20 bp homologous to the linearized pBD-GAL4-Cam vector at its 5′-end. Two separate PCRs were carried out, each containing one flanking primer and one mutagenic primer. The two PCR products were purified from a 1% agarose gel after DNA electrophoresis, mixed together in an equal molar ratio, and assembled by a primerless PCR. The assembled products were amplified with the two flanking primers. The resulting PCR product was purified from the gel and cotransformed into yeast YRG-2 cells with the EcoRI-SalI-digested pBD-GAL4-Cam vector to obtain circular plasmids. Plasmids were rescued from yeast using the Zymoprep kit (Zymo Research Corp.) and transformed into E. coli DH5α to obtain plasmid DNA with high purity. For single mutants, the wild type LBD gene was used as the template for the overlap extension PCR, while for double and triple mutants, the corresponding single and double mutant LBD genes were used as the template, respectively. DNA sequencing was carried out to confirm the presence of the introduced specific mutations and the absence of PCR-associated random mutations. The sequences of the primers will be provided upon request.DNA Shuffling and DNA Sequencing—DNA shuffling of the second round hERα variants was carried out as described elsewhere (17Zhao H. Arnold F.H. Nucleic Acids Res. 1997; 25: 1307-1308Crossref PubMed Scopus (190) Google Scholar). DNA sequencing of the evolved hERα variants was carried out as described in Ref. 18Chen Z. Zhao H. Gene (Amst.). 2003; 306: 127-134Crossref PubMed Scopus (12) Google Scholar.Cell Culture and Transfection—Cell culture and transfection were performed as described elsewhere (19Muthyala R.S. Sheng S.B. Carlson K.E. Katzenellenbogen B.S. Katzenellenbogen J.A. J. Med. Chem. 2003; 46: 1589-1602Crossref PubMed Scopus (83) Google Scholar).Hormone Binding Assays—E. coli BL21(DE3) cells transformed with the pET15b plasmid containing the wild type or mutant hERα LBD fragments encoding residues 312–595 were grown at 37 °C until A600 = 0.6. Protein expression was then induced with 0.5 mm isopropyl-β-d-thiogalactopyranoside at 25 °C for 5–7 h. Cells were harvested by centrifugation, resuspended in buffer B (50 mm Tris buffer, pH 8.0, 10% glycerol, and 10 mm β-mercaptoethanol) at 10 ml/g of wet cells, and lysed by a French press according to the standard protocol suggested by the manufacturer. The cell debris were separated from the supernatants by centrifugation for 30 min at 10,000 × g. The resulting supernatants were used in all hormone binding assays.The equilibrium ligand binding assay was performed essentially as described elsewhere (19Muthyala R.S. Sheng S.B. Carlson K.E. Katzenellenbogen B.S. Katzenellenbogen J.A. J. Med. Chem. 2003; 46: 1589-1602Crossref PubMed Scopus (83) Google Scholar) except that E. coli cell extracts rather than purified proteins were used in the assays. Relative binding affinity (RBA) measurements were determined as previously reported (20Katzenellenbogen J.A. Johnson Jr., H.J. Myers H.N. Biochemistry. 1973; 12: 4085-4092Crossref PubMed Scopus (257) Google Scholar) with slight modifications. The cell extracts containing wild type or mutant hERα were diluted to 1 nm in buffer B and incubated with buffer alone or with several concentrations of unlabeled competitor together with 2 or 10 nm [3H]E2 at 0 °C for 16–24 h. At the end of the incubation, the solutions were processed the same way as in the equilibrium ligand binding assay. The unlabeled competitors were diluted to a 1:1 ratio of dimethylformamide:buffer B to ensure solubility, and the final concentration of dimethylformamide in the solution was 7%.Molecular Modeling—The hERα LBD crystal structure (Protein Data Bank code 1GWR) was imported into MOE (Molecular Operating Environment, Chemical Computing Group Inc., Montreal, Quebec, Canada). Force field MMFF94s was used for all simulations. After fixing all missing residues and atoms manually, hydrogen atoms were added. Two steps of energy minimization were performed, first with all the α-carbons fixed and then without constraints. Residue Glu-353 was mutated to Gln using the MOE Rotamer Explorer function, and the rotamer that adopts a similar conformation as the original Glu was selected. Testosterone was built using the MOE Builder function and energy-minimized. This testosterone structure was superimposable with the DHT structure found in the rat AR LBD crystal structure complexed with DHT (Protein Data Bank code 1I37). Testosterone was manually placed into the ligand binding pocket of the mutated hERα (E353Q) followed by running the docking program (initial temperature, 5000 K; iteration limit, 8000 cycles; 10 cycles per run; 25 runs). The lowest energy docked conformation was further energy-minimized with atoms more than 10 Å away from the ligand being fixed. A short molecular dynamic simulation was then carried out to release all the energy constraints caused by ligand docking. During the molecular dynamic simulation, the energy-minimized receptor-ligand complex was heated to 310 K at 100 K/ps and equilibrated at 310 K for 20 ps with time steps of 1 fs and atoms more than 10Å away from the ligand being fixed. For the structural comparison between AR and ER, the structures of rat AR (Protein Data Bank code 1I37) 2Since the protein sequences for rat and human AR LBD are identical, the affinity value and crystal structure of the rat AR were used for the human AR in this report. and hERα (Protein Data Bank 1GWR) were imported into MOE and super-imposed based on sequence homology.RESULTSLibrary Screening Using Yeast Two-hybrid System—To isolate human ERα variants with altered transcriptional activation (transactivation) activity, we developed an efficient and sensitive high throughput screening method based on the yeast two-hybrid system. In this method, the cDNA encoding hERα amino acids 312–595 containing most of the LBD domain (hERα amino acids 303–553) and the F domain (hERα amino acids 554–595) was fused to the gene encoding the GAL4 DNA binding domain in plasmid pBD-GAL4-Cam (Stratagene) to form the “bait plasmid” pBD-GAL4 hERα, and the gene encoding human SRC-1 was fused to the gene encoding the GAL4 activation domain in plasmid pGAD424 (Clontech) to form the “prey plasmid” pGAD424 SRC-1.Both plasmids were transformed and expressed in S. cerevisiae YRG-2, which contains a GAL4-regulated HIS3 reporter construct on its chromosome. The HIS3 reporter provides strong nutritional selection (only cells expressing HIS3 gene product, which is an essential enzyme in the histidine biosynthesis pathway, can grow in a minimal medium lacking histidine). In the presence of agonistic ligands, the LBD undergoes a conformational change and binds to SRC-1, which brings the GAL4 DNA binding domain and the GAL4 activation domain in proximity, thus activating the transcription of the reporter gene. In general, the cell growth rate is proportional to the strength of the ligand-receptor interaction. In the absence of agonistic ligands, no transcription of reporter genes will occur. The functional interaction of ERα LBD with the coactivator SRC-1 is critical for effecting transcription in mammalian cells (5Aranda A. Pascual A. Physiol. Rev. 2001; 81: 1269-1304Crossref PubMed Scopus (1162) Google Scholar).We have validated this yeast two-hybrid-based selection/screening method using the wild type hERα LBD in yeast cells. All experiments were done in the 96-well plates where A600 (cell density) was measured. First, it was shown that cells bearing plasmids pBD-GAL4 hERα and pGAD424 SRC-1 responded to E2 at a subnanomolar concentration, while cells bearing plasmid pBD-GAL4 ERα alone had no response to E2 up to a micromolar concentration, indicating that the cell growth assay is tripartite (Fig. 2A). Second, it was found that the ability of cells bearing plasmids pBD-GAL4 hERα and pGAD424 SRC-1 to activate transcription in response to a ligand generally correlated with the RBA of the ligand (E2 (100) > 17α-estradiol (58) > 2-OH-estradiol (7) > testosterone (<0.01) > progesterone (<0.001), the RBA values (in parentheses) were taken from Ref. 21Kuiper G.G. Carlsson B. Grandien K. Enmark E. Haggblad J. Nilsson S. Gustafsson J.A. Endocrinology. 1997; 138: 863-870Crossref PubMed Scopus (3678) Google Scholar) with greater response seen with ligands with higher RBAs (Fig. 2B). These results indicate this selection/screening method is very sensitive and specific. It should be noted that the half-maximal effect concentration (EC50, the ligand concentration that causes a half-maximal response) of the wild type hERα LBD for E2 was estimated to be ∼0.6 nm, which is in good agreement with the reported value of the full-length hERα for E2 binding (12Wrenn C.K. Katzenellenbogen B.S. J. Biol. Chem. 1993; 268: 24089-24098Abstract Full Text PDF PubMed Google Scholar).Fig. 2Experiments designed to validate the yeast two-hybrid-based high throughput screening method.A, comparison of dose responses of YRG2 cells bearing pBD-GAL4 hERα and pGAD424 SRC-1 and YRG2 cells bearing plasmid pBD-GAL4 hERα alone. B, comparison of dose responses of a panel of diverse ligands to the wild type hERα.View Large Image Figure ViewerDownload Hi-res image Download (PPT)For library screening, we used a two-tiered strategy consisting of an agar plate-based selection followed by a 96-well platebased screening. In the selection method, the mutagenized hERα LBD variants were co-transformed with pGAD424 SRC-1 into S. cerevisiae YRG2 and plated on an agar plate containing minimum medium lacking tryptophan, leucine, and histidine and supplemented with a predefined testosterone concentration. To eliminate the false positives caused by mutations resulting in ligand-independent responses, the colonies that appeared on the agar plate were picked with toothpicks and restreaked onto two agar plates, one with testosterone and the other without testosterone. The colonies appearing on the agar plate with testosterone but not on the agar plate without testosterone were picked and assayed in the 96-well plate to determine their EC50 values. Moreover, to ensure that the improved EC50 value of a positive variant was plasmid-linked, the plasmid was rescued from the corresponding variant and transformed into fresh YRG-2 cells to confirm that the same EC50 value could be obtained.Directed Evolution of hERαLBD Variants with Increased Response to Testosterone—In the first round of directed evolution, we used error-prone PCR to introduce random point mutations (one to two amino acid substitutions per gene on average) into the LBD fragment consisting of hERα amino acids 312–595. Transformed yeast cells bearing a library of hERα LBD variants (∼60,000) were plated on minimum medium lacking tryptophan, leucine, and histidine and supplemented with 5 × 10-7m testosterone. Cells bearing the wild type ERα LBD were used as a negative control. Fifty-three colonies appearing on the selection plates were picked and streaked on two plates, one with testosterone and the other without testosterone. Three ligand-dependent clones were identified and selected for a quantitative dose-response measurement in 96-well plates in which the cell densities were determined over a range of testosterone concentrations.Two clones (T7 and T17) showed increased ligand sensitivity and were selected for the second round of directed evolution. Two independent libraries of mutants were created using error-prone PCR. Using the same screening strategy, ∼120,000 c" @default.
• W2045128521 created "2016-06-24" @default.
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• W2045128521 date "2004-08-01" @default.
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• W2045128521 title "Directed Evolution of Human Estrogen Receptor Variants with Significantly Enhanced Androgen Specificity and Affinity" @default.
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• W2045128521 doi "https://doi.org/10.1074/jbc.m402118200" @default.
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