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Different Residues of the Human Estrogen Receptor Are Involved in the Recognition of Structurally Diverse Estrogens and Antiestrogens

1997; Elsevier BV; Volume: 272; Issue: 8 Linguagem: Inglês

10.1074/jbc.272.8.5069

1083-351X

Kirk Ekena, Karen E. Weis, John A. Katzenellenbogen, Benita S. Katzenellenbogen,

Computational Drug Discovery Methods

We have previously examined, by alanine scanning mutagenesis, amino acids 515-535 of the estrogen receptor (ER) ligand binding domain to determine which of these residues are important in estradiol binding. Mutation at four sites that potentially lie along one face of an α-helix, Gly521, His524, Leu525, and Met528, all significantly impaired estradiol binding by the ER (Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 20053-20059). In this report, we compare the pattern of residues that are important in the recognition of several structurally diverse estrogen agonists and antagonists (the synthetic nonsteroidal agonist hexestrol, an agonist derived from the mold metabolite zearalenone, P1496, and the partial agonist-antagonist trans-hydroxytamoxifen) with those that are predicted to contact estradiol in the receptor-ligand complex. Although there are some similarities in the pattern of residue recognition among all four ligands, each ligand showed distinct differences as well. Interestingly, alanine substitution at only one residue, the leucine at position 525, was found to inhibit binding of all the ligands tested. Another residue, His524, was found to be important in the recognition of three different agonists but not trans-hydroxytamoxifen (the only ligand lacking a second hydroxyl group). The recognition of estradiol and another agonist, P1496, was impaired by the G521A mutation, whereas ligand-induced activity by the two compounds that lack B- and C-rings, hexestrol and trans-hydroxytamoxifen, was unaffected. Our findings demonstrate that these ligands fit into the ER ligand binding pocket differently and that each contacts a distinct set of amino acids. The smaller ligands (estradiol and hexestrol) have a narrower footprint of interacting residues than the larger ligands (P1496 and trans-hydroxytamoxifen). This pattern of interaction is most consistent with the amino acids within this region being in contact with the portion of these ligands that corresponds to the D-ring end of estradiol. The interplay between the shape of an ER ligand and the residues that support its binding to ER may potentially underlie the selective actions of different ER ligands in various cell and promoter contexts. We have previously examined, by alanine scanning mutagenesis, amino acids 515-535 of the estrogen receptor (ER) ligand binding domain to determine which of these residues are important in estradiol binding. Mutation at four sites that potentially lie along one face of an α-helix, Gly521, His524, Leu525, and Met528, all significantly impaired estradiol binding by the ER (Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 20053-20059). In this report, we compare the pattern of residues that are important in the recognition of several structurally diverse estrogen agonists and antagonists (the synthetic nonsteroidal agonist hexestrol, an agonist derived from the mold metabolite zearalenone, P1496, and the partial agonist-antagonist trans-hydroxytamoxifen) with those that are predicted to contact estradiol in the receptor-ligand complex. Although there are some similarities in the pattern of residue recognition among all four ligands, each ligand showed distinct differences as well. Interestingly, alanine substitution at only one residue, the leucine at position 525, was found to inhibit binding of all the ligands tested. Another residue, His524, was found to be important in the recognition of three different agonists but not trans-hydroxytamoxifen (the only ligand lacking a second hydroxyl group). The recognition of estradiol and another agonist, P1496, was impaired by the G521A mutation, whereas ligand-induced activity by the two compounds that lack B- and C-rings, hexestrol and trans-hydroxytamoxifen, was unaffected. Our findings demonstrate that these ligands fit into the ER ligand binding pocket differently and that each contacts a distinct set of amino acids. The smaller ligands (estradiol and hexestrol) have a narrower footprint of interacting residues than the larger ligands (P1496 and trans-hydroxytamoxifen). This pattern of interaction is most consistent with the amino acids within this region being in contact with the portion of these ligands that corresponds to the D-ring end of estradiol. The interplay between the shape of an ER ligand and the residues that support its binding to ER may potentially underlie the selective actions of different ER ligands in various cell and promoter contexts. INTRODUCTIONEstrogens strongly influence the growth, differentiation, and functioning of the reproductive system, including the mammary gland and the uterus (1Katzenellenbogen B.S. Biol. Reprod. 1996; 54: 287-293Google Scholar, 2Sarrel P.M. Lufkin E.G. Oursler M.J. Keefe D. Sci. Amer. Sci. Med. 1994; 1: 44-53Google Scholar). The effect of estrogen on the mammary gland is of particular interest because of its link to breast cancer; the proliferation and metastatic activity of nearly 40% of breast cancers are stimulated by estrogens (3Read L.D. Katzenellenbogen B.S. Dickson R.B. Lippman M.E. Genes, Oncogenes, and Hormones: Advances in Cellular and Molecular Biology of Breast Cancer. Kluwer Academic Publishers, Boston1991: 277-299Google Scholar).Estrogens exert their effects by acting through a ligand-activated transcription factor, the estrogen receptor (ER). 1The abbreviations used are: ERestrogen receptorCATchloramphenicol acetyltransferaseCMVcytomegalovirusEREestrogen response elementICIICI 164,384RAretinoic acidRARretinoic acid receptorT3thyroid hormoneTOTtrans-hydroxytamoxifenWTwild type. A member of a superfamily of nuclear receptors, the estrogen receptor contains a highly conserved DNA-binding domain, domain C, and a highly conserved ligand-binding domain, domain E. In addition to its ligand binding activity, the E domain also possesses dimerization activity and a hormone-dependent activation function (AF-2). A hormone-independent activation function (AF-1) is located within the receptor's A/B domain. In the presence of ligand, the ER bound to an estrogen response element (ERE) can either activate or suppress transcription of a downstream target gene in a cell- and promoter-specific manner (4Evans R.M. Science. 1988; 240: 889-895Google Scholar–11Fujimoto N. Katzenellenbogen B.S. Mol. Endocrinol. 1994; 8: 296-304Google Scholar).Estrogen antagonists such as tamoxifen have been successfully used in the treatment of ER-positive breast cancers (3Read L.D. Katzenellenbogen B.S. Dickson R.B. Lippman M.E. Genes, Oncogenes, and Hormones: Advances in Cellular and Molecular Biology of Breast Cancer. Kluwer Academic Publishers, Boston1991: 277-299Google Scholar, 12Wakeling A.E. Biochem. Pharmacol. 1995; 49: 1545-1549Google Scholar, 13Wakeling A.E. Pasqualini J.R. Katzenellenbogen B.S. Hormone-Dependent Cancer. Marcel Dekker Publishers, New York1996: 107-118Google Scholar). These antagonists compete with estradiol for binding to the receptor but fail to elicit transcriptional activity. Unfortunately, tamoxifen treatment has several drawbacks. First, tamoxifen is not a complete antagonist and has agonistic effects in certain cell and promoter contexts. Second, breast cancer cells often become resistant to this treatment (14Katzenellenbogen B.S. J. Natl. Cancer Inst. 1991; 83: 1434-1435Google Scholar). Finally, despite the need to inhibit ER activity in breast cancer cells, the retention of some ER activity is important in a number of other biological processes, including bone maintenance, the cardiovascular system, and liver metabolism (2Sarrel P.M. Lufkin E.G. Oursler M.J. Keefe D. Sci. Amer. Sci. Med. 1994; 1: 44-53Google Scholar). Thus, it becomes important to understand how the ER recognizes and interprets ligands of different structure, that is, which amino acids are involved in their binding, and thereby how these ligands might result in different activities in various cell types. Such information could then be used to design new estrogen agonists and antagonists with target cell- and response-selective biological effects.We and others have identified regions of the ER involved in recognition of ligand (15Harlow K.W. Smith D.N. Katzenellenbogen J.A. Greene G.L. Katzenellenbogen B.S. J. Biol. Chem. 1989; 264: 17476-17485Google Scholar–18Danielian P.S. White R. Hoare S.A. Fawell S.E. Parker M.G. Mol. Endocrinol. 1993; 7: 232-240Google Scholar). We have found amino acids near Cys530 to be important in estradiol binding, whereas residues C-terminal to position 535 have been shown to contain AF-2 activity (19Wrenn C.K. Katzenellenbogen B.S. J. Biol. Chem. 1993; 268: 24089-24098Google Scholar–21Danielian P.S. White R. Lees J.A. Parker M.G. EMBO J. 1992; 11: 1025-1033Google Scholar), and residues 507-514 may contain dimerization activity (20Fawell S.E. Lees J.A. White R. Parker M.G. Cell. 1990; 60: 953-962Google Scholar, 22Lees J.A. Fawell S.E. White R. Parker M.G. Mol. Cell. Biol. 1990; 10: 5529-5531Google Scholar). Recently, we used alanine scanning mutagenesis to identify those amino acids in the 515-535 region of the ER ligand binding domain important in estradiol binding (23Ekena K. Weis K.E. Katzenellenbogen J.A. Katzenellenbogen B.S. J. Biol. Chem. 1996; 271: 20053-20059Google Scholar). Mutation of the amino acids Gly521, His524, Leu525, and Met528 to alanine affected the ability of the ER to bind estradiol and had a parallel effect on estrogen-dependent transcription without affecting the overall activity of the receptor. These results, taken together with structural predictions based on crystal structures of other ligand-bound nuclear receptors, indicated that these four amino acids of the ER may lie along one face of an α-helix and are presumed to be in contact with the bound estradiol.In this report, we extend those studies to include four other estrogen agonists and antagonists with varied structures: the synthetic nonsteroidal agonist meso-hexestrol; P1496, an agonist derived from the mold metabolite zearalenone; the partial agonist-antagonist trans-hydroxytamoxifen; and the pure antagonist ICI. Interestingly, we observe that different amino acids in the 515-535 region of the ER are important in the recognition of these different ligands. Only one amino acid (Leu525) was found to be important in binding all of the ligands. By comparing the amino acids important in binding each of the ligands and comparing the ER amino acid sequence with other nuclear receptors and published crystal structures, we suggest which portion of these ligands are in contact with this region of the ER ligand binding pocket.DISCUSSIONWe have probed the interaction that various ligands have with a portion of the ligand binding domain of the human estrogen receptor (ER) by alanine scanning mutagenesis, studying in addition to estradiol the behavior of three high affinity ligands: two nonsteroidal agonists, the simple, symmetrical synthetic ligand meso-hexestrol, and P1496, a derivative of the fungal produced estrogen zearalenone, as well as the partial antagonist, trans-hydroxytamoxifen, the potent metabolite of tamoxifen. As in our earlier study with estradiol, we found that the substitution of certain residues in this ER region to alanine caused a significant to marked reduction in the potency of these ligands in inducing transcriptional activity. In addition, the principal finding from this study is that each of the ligands displayed a distinct footprint of interacting residues in the alanine scanning mutagenesis, indicating that the structural differences in these ligands result in different patterns of interaction with amino acids of the ER (Table I).We have confirmed that the alterations in ligand-induced transcriptional activity of the mutant receptors correspond to a dose shift (i.e. change in potency) in the transactivation response. Previously, with estradiol, we established that the decrease in transcription activation potency correlated well with a decreased binding affinity of estradiol to the mutant receptors (23Ekena K. Weis K.E. Katzenellenbogen J.A. Katzenellenbogen B.S. J. Biol. Chem. 1996; 271: 20053-20059Google Scholar). Thus, these interacting residues are considered to be ones that are important in determining the binding affinity of these ligands.The effects of mutational change at certain sites were of particular interest. First, the L525A mutation was the only mutation that strongly affected the transcriptional potency of all four ligands (Table I). However, P1496, the bulkiest of the ligands, was least affected, possibly because it may best be able to compensate for the reduced bulk in ER that results from the Leu to Ala mutation. Second, the H524A mutation had a very strong effect on hexestrol and P1496-induced ER activity and a major effect on estradiol but no effect on TOT. It is of note that TOT is the only ligand that lacks a second hydroxyl group with which this histidine may be interacting. Third, the G521A substitution strongly affected estradiol and P1496 transactivation yet had only a modest effect on TOT and no effect on hexestrol. As discussed later, the smaller size of certain portions of the hexestrol and TOT structures, compared with estradiol and P1496, might enable them to tolerate the increased residue size resulting from the G521A substitution. Fourth, the K520A, M522A, N532A, and P535A substitutions had an effect on TOT induced transactivation but did so by reducing the maximal transcriptional activity of the mutant ER-TOT complex rather than by shifting the dose response curve. This decreased transactivation potential suggests that these residues do not involve the binding interaction between TOT and ER but rather affect the manner in which the liganded complex interacts with other components important for activating transcription. It is interesting that certain specific contacts between TOT and ER appear to affect only its balance of agonist and antagonist activity (such as residues 520, 522, and 535), whereas others affect only its binding affinity (such as residues 515, 516, and 525).From our previous study on the effect of alanine scanning mutagenesis on the binding of estradiol and ligand-induced transactivation of the ER (23Ekena K. Weis K.E. Katzenellenbogen J.A. Katzenellenbogen B.S. J. Biol. Chem. 1996; 271: 20053-20059Google Scholar), we showed that the residues most affected by alanine substitution, Gly521, His524, Leu525, and Met528, could be displayed on three adjacent turns on one face of an α-helix. By sequence comparison with the human retinoic acid receptor-γ (RAR) and the rat thyroid hormone receptor-α1 (TR), these sites lie on the inner face of helix-11 and correspond to residues in the RAR-retinoic acid (RA) and TR-thyroid hormone (T3) structures that are in close contact with the bound ligands (32Renaud J.-P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Google Scholar, 33Wagner R.L. Apriletti J.W. McGrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Google Scholar). In the case of RAR-RA, these residues are in contact with the apolar end of the ligand, the β-ionone portion, and in the case of TR-T3, these residues contact the distal phenolic ring, with His381 in TR (corresponding to Gly521 in ER), making a hydrogen bond to the distal phenolic hydroxyl group.In this study, we find that the pattern of residues in the 515-535 region of ER that affect ligand-induced transcription differs depending on the structure of the activating ligand. A comparison of the structures of these ligands (Fig. 1) together with their interacting residues (Table I) allows one to formulate a model for the basic orientation of these ligands within the binding cavity of the ER (Fig. 6). By analogy with the TR-T3 and RAR-RA structures, this model shows the 515-535 region of ER as a helical region (corresponding to a portion of helix-10 and all of helix-11) and the beginning of the loop region between helix-11 and −12. The residues that affect the transcriptional potency of each of the four ligands are indicated schematically with circles. The helix and loop of ER and the structures of the ligands are presented on the same dimensional scale to illustrate the differential interaction that these four ligands have with the ER.The four activating ligands, estradiol, hexestrol, P1496, and TOT, all have one common structural feature, a phenol, yet they show different patterns of residues whose substitution with alanine affects their transcriptional potency. These differences suggest that the ligands are not making contact with the 515-535 portion of the ER through their structurally common A-ring like feature. The rather narrow footprint of residues that affect hexestrol recognition (principally two residues, His524 and Leu525, on one turn of the helix) suggests that contact is being made with a portion of the hexestrol structure that is narrower than the corresponding region in estradiol (whose potency is affected principally by three residues, Gly521, His524, and Leu525, on two helical turns). In contrast, the much wider footprint of residues that affect P1496 binding (four or more residues on at least three turns) suggests that their interaction is with a region of the ligand that is larger than the corresponding region in estradiol. Thus, if the phenol rings of these three ligands are oriented similarly and away from the 515-535 region, then it is the D-ring end of estradiol and the corresponding narrower region of hexestrol and the wider region of P1496 that are likely to be in contact with this region of the ER.The pattern of residues that affect the transcriptional potency of TOT is rather unique. The interaction of TOT with the middle of the helical region is limited. In particular, its lack of interaction with His524 suggests that in lacking a second hydroxyl group, TOT is indifferent to the hydrogen bonding characteristics of the residue at this site. On the other hand, TOT is the only ligand whose transcriptional potency is affected by residues near the N terminus of the examined region (Arg515 and His516), suggesting that its side chain may extend to be in contact with this region of the receptor.Altogether, the comparison of ligand structure with the pattern of residues where mutation affects their transcriptional potency suggests that selected residues in the 515-535 region of the receptor are in contact with a portion of these ligands that corresponds to the D-ring end of estradiol. Thus, a rough comparison can be made between the orientation of these estrogens in ER and the RA and T3 ligands in RAR and TR, respectively. It is the D-ring end of estradiol that corresponds to the apolar end of RA and the phenol of T3 that is in contact with the helix-11 region of these nuclear receptors. The A-ring or phenolic portion of the estrogen ligands, which corresponds to the polar end of the RA and T3 ligands, is directed away from helix-11.It is well recognized now that the ER and related nuclear receptors show transcriptional activity that is modulated by the nature of the particular gene promoter and the cellular background, consistent with ligand-receptor interaction with cell- and promoter-specific factors and transcriptional coactivators (5Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Google Scholar, 34Katzenellenbogen J.A. O'Malley B.W. Katzenellenbogen B.S. Mol. Endocrinol. 1996; 10: 119-131Google Scholar). The interplay between the shape of an ER ligand and the residues that support its binding to ER, as studied here, may potentially underlie the selective actions of different ER ligands in various target cells and promoter contexts. INTRODUCTIONEstrogens strongly influence the growth, differentiation, and functioning of the reproductive system, including the mammary gland and the uterus (1Katzenellenbogen B.S. Biol. Reprod. 1996; 54: 287-293Google Scholar, 2Sarrel P.M. Lufkin E.G. Oursler M.J. Keefe D. Sci. Amer. Sci. Med. 1994; 1: 44-53Google Scholar). The effect of estrogen on the mammary gland is of particular interest because of its link to breast cancer; the proliferation and metastatic activity of nearly 40% of breast cancers are stimulated by estrogens (3Read L.D. Katzenellenbogen B.S. Dickson R.B. Lippman M.E. Genes, Oncogenes, and Hormones: Advances in Cellular and Molecular Biology of Breast Cancer. Kluwer Academic Publishers, Boston1991: 277-299Google Scholar).Estrogens exert their effects by acting through a ligand-activated transcription factor, the estrogen receptor (ER). 1The abbreviations used are: ERestrogen receptorCATchloramphenicol acetyltransferaseCMVcytomegalovirusEREestrogen response elementICIICI 164,384RAretinoic acidRARretinoic acid receptorT3thyroid hormoneTOTtrans-hydroxytamoxifenWTwild type. A member of a superfamily of nuclear receptors, the estrogen receptor contains a highly conserved DNA-binding domain, domain C, and a highly conserved ligand-binding domain, domain E. In addition to its ligand binding activity, the E domain also possesses dimerization activity and a hormone-dependent activation function (AF-2). A hormone-independent activation function (AF-1) is located within the receptor's A/B domain. In the presence of ligand, the ER bound to an estrogen response element (ERE) can either activate or suppress transcription of a downstream target gene in a cell- and promoter-specific manner (4Evans R.M. Science. 1988; 240: 889-895Google Scholar–11Fujimoto N. Katzenellenbogen B.S. Mol. Endocrinol. 1994; 8: 296-304Google Scholar).Estrogen antagonists such as tamoxifen have been successfully used in the treatment of ER-positive breast cancers (3Read L.D. Katzenellenbogen B.S. Dickson R.B. Lippman M.E. Genes, Oncogenes, and Hormones: Advances in Cellular and Molecular Biology of Breast Cancer. Kluwer Academic Publishers, Boston1991: 277-299Google Scholar, 12Wakeling A.E. Biochem. Pharmacol. 1995; 49: 1545-1549Google Scholar, 13Wakeling A.E. Pasqualini J.R. Katzenellenbogen B.S. Hormone-Dependent Cancer. Marcel Dekker Publishers, New York1996: 107-118Google Scholar). These antagonists compete with estradiol for binding to the receptor but fail to elicit transcriptional activity. Unfortunately, tamoxifen treatment has several drawbacks. First, tamoxifen is not a complete antagonist and has agonistic effects in certain cell and promoter contexts. Second, breast cancer cells often become resistant to this treatment (14Katzenellenbogen B.S. J. Natl. Cancer Inst. 1991; 83: 1434-1435Google Scholar). Finally, despite the need to inhibit ER activity in breast cancer cells, the retention of some ER activity is important in a number of other biological processes, including bone maintenance, the cardiovascular system, and liver metabolism (2Sarrel P.M. Lufkin E.G. Oursler M.J. Keefe D. Sci. Amer. Sci. Med. 1994; 1: 44-53Google Scholar). Thus, it becomes important to understand how the ER recognizes and interprets ligands of different structure, that is, which amino acids are involved in their binding, and thereby how these ligands might result in different activities in various cell types. Such information could then be used to design new estrogen agonists and antagonists with target cell- and response-selective biological effects.We and others have identified regions of the ER involved in recognition of ligand (15Harlow K.W. Smith D.N. Katzenellenbogen J.A. Greene G.L. Katzenellenbogen B.S. J. Biol. Chem. 1989; 264: 17476-17485Google Scholar–18Danielian P.S. White R. Hoare S.A. Fawell S.E. Parker M.G. Mol. Endocrinol. 1993; 7: 232-240Google Scholar). We have found amino acids near Cys530 to be important in estradiol binding, whereas residues C-terminal to position 535 have been shown to contain AF-2 activity (19Wrenn C.K. Katzenellenbogen B.S. J. Biol. Chem. 1993; 268: 24089-24098Google Scholar–21Danielian P.S. White R. Lees J.A. Parker M.G. EMBO J. 1992; 11: 1025-1033Google Scholar), and residues 507-514 may contain dimerization activity (20Fawell S.E. Lees J.A. White R. Parker M.G. Cell. 1990; 60: 953-962Google Scholar, 22Lees J.A. Fawell S.E. White R. Parker M.G. Mol. Cell. Biol. 1990; 10: 5529-5531Google Scholar). Recently, we used alanine scanning mutagenesis to identify those amino acids in the 515-535 region of the ER ligand binding domain important in estradiol binding (23Ekena K. Weis K.E. Katzenellenbogen J.A. Katzenellenbogen B.S. J. Biol. Chem. 1996; 271: 20053-20059Google Scholar). Mutation of the amino acids Gly521, His524, Leu525, and Met528 to alanine affected the ability of the ER to bind estradiol and had a parallel effect on estrogen-dependent transcription without affecting the overall activity of the receptor. These results, taken together with structural predictions based on crystal structures of other ligand-bound nuclear receptors, indicated that these four amino acids of the ER may lie along one face of an α-helix and are presumed to be in contact with the bound estradiol.In this report, we extend those studies to include four other estrogen agonists and antagonists with varied structures: the synthetic nonsteroidal agonist meso-hexestrol; P1496, an agonist derived from the mold metabolite zearalenone; the partial agonist-antagonist trans-hydroxytamoxifen; and the pure antagonist ICI. Interestingly, we observe that different amino acids in the 515-535 region of the ER are important in the recognition of these different ligands. Only one amino acid (Leu525) was found to be important in binding all of the ligands. By comparing the amino acids important in binding each of the ligands and comparing the ER amino acid sequence with other nuclear receptors and published crystal structures, we suggest which portion of these ligands are in contact with this region of the ER ligand binding pocket.