I. Estrogen Receptor Structure-Function Overview

Written by Ms. Suparna Saha for "Independent Study" (03-410; Spring, 2001), the Overview describes many of the key features of the estrogen receptor (ER) and its modes of action. This section is intended to provide sufficient background for additional reading (See Selected References.); browsing (See Links to ER on the Web.); and structure viewing (Chime pages, linked below).

II. Estrogen Receptor Structure Models

Links to Chime pages based on the structure papers read in the "Independent Study" course. The displays emphasize the ligand (and DNA site) specificity and the structural differences between agonist and antagonist complexes reported by crystallographers.

I. Estrogen Receptor Structure-Function Overview

All nuclear receptors have a hydrophobic pocket into which its specific ligand binds, with helix 12 (H12) being the key response element of NR’s. When an agonist is bound to a NR, H12 is oriented anti-parallel to H11, capping the ligand binding pocket. This leaves a hydrophobic groove exposed for the binding of coregulator proteins. When an antagonist is bound, H12 is displaced via an extended side chain. H12 moves outward, rotates, and packs into the hydrophobic groove between helices 3, 4, and 5. As a result, coactivators needed for transcription cannot bind. The following two images show ERa with an agonist (left) and antagonist (right) bound to it.

Estrogen Receptor Ligand-Binding Domain Complexed to Estradiol	Estrogen Receptor Ligand-Binding Domain Complexed to Raloxifene

H12 is shown as a magenta coil in the "green sububits". The other subunit in each structure is displayed as "Cartoons" with "Structure" coloring (RasMol). The ligands are Spacefill.
Helix 12 Slide Show displays JPEG views of several other ER-ligand complexes superimposed on ER-EST. References to the structure papers are listed.

Two Different Estrogen Receptors
Recent studies have revealed the existence of two distinct estrogen receptors in our bodies: ERa and ERb. While they both bind estrogen as well as other agonists and antagonists, the two receptors have distinctly different localizations and concentrations within our body. Structural differences also exist between the two. allowing for a wide range of diverse and complex processes to take place. The following diagram, adapted from the Gustafsson review (1999), shows the distribution of ERa and ERb

Structural Differences between ERa and ERb
Two of the most interesting sites on the ER molecule are its ligand binding domain (LBD), otherwise known as AF-2, and its growth factor binding domain, otherwise known as AF-1. In addition, the DNA-binding domain (DBD) is responsible for binding at estrogen response elements (ERE) on the chromosome. A subtle difference between the two in their ligand-binding pockets is the substituion of Leu 338 in ERa with Met 384 in ERb.

The following diagram, adapted from the Gustafsson review (1999), compares the amino acid sequences of the two receptors:

The separate domains are identified in the ERa diagram; the numbers in the ERb diagram show the sequence identity as %.

Functional Differences
Interestingly, ERa and ERb, when complexed with estrogen, were shown to signal in opposite ways from an AP1 site, with estrogen activating transcription in the presence of ERa and inhibiting transcription in the presence of ERb. The ER ligands tamoxifen, raloxifene, and ICI-164384 were activators with ERb as well as ERa, although the degree of agonism differed between cell types. These molecules are examples of SERM’s, selective estrogen receptor modulators. Thus, the role of estrogen complexed to ERb appears to be to turn off transcription of these genes, whereas the SERMs may override this blockade and activate gene transcription.

Effects of SERM's
Most recent drugs targeted to the ER, such as tamoxifen, ICI-164384, and raloxifene act as either ER antagonists or agonists depending on the species, tissue, and the dose administered. For instance, raloxifene has been reported to act as an antiestrogen in breast tumor tissue and the brain, while it has potentially beneficial estrogen-like effects in bone and in modulating factors associated with cardio-vascular diseases. Another example is tamoxifen, which was developed as an antiestrogen for the treatment of breast cancer and was subsequently shown to have estrogen-like effects on bone and the cardiovascular system. However, the potentially beneficial effects of tamoxifen in reducing the risk of osteoporotic fractures and coronary heart disease in postmenopausal women are at least partially offset by its estrogenic effects on the uterus, increasing risk of endometrial cancer development.

Binding Assays
Typical assays to gauge the binding affinities of estrogen analogs or new drugs follow a standard pattern. The receptors are infused with E2, and a binding curve is obtained based on varying concentrations of E2 present. Then, the molecule in question is added in varying concentrations, acting as a competitive inhibitor, and its ability to bind is plotted against E2's. Most of these assays are depicted using Scatchard plots. The relative binding constants of some well-studied molecules are as follows:

Ligand	ERa	ERb
17b-Estradiol ("E2" or "EST")	100	100
Diethylstilbestrol	468	295
Tamoxifen	6	7

The following chart compares the binding of reservatrol to that of estrogen. The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, it was thought to be a phytoestrogen. The following figure was taken from an article by Gehm, et al. (1997):

Estrogen receptor binding assays were performed by using 0.1 nM (circles), 0.3 nM (triangles), or 1.0 nM (squares). ¹²⁵I-estradiol competed with the indicated concentrations of resveratrol (open symbols) or unlabeled estradiol (filled circles). Each point represents the mean and range of duplicate assays after subtraction of nonspecific binding. All results are shown as percentage of binding in the absence of competitor.

As can be seen from the graph, estradiol has a much higher binding affinity for ER than does resveratrol. It is important to note that binding affninties in vitro are often much higher than those observed in vivo due to the numerous other pathways and cellular processes that may interfere with binding.

Coactivators and Corepressors
Transactivation requires the recruitment of coactivators, such as SRC-1, that posess histone acetyltransferase activity or can recruit a histone acetyl transferase. This complex can decompact the chromatin, enabling a transcription initiation complex to form. Silencing involves the recruitment of corepressors, such as SMRT, and histone deacetyltransferases.

The transconformation of H12, along with other associated structural changes, creates a surface on the receptor that can bind coactivators such as SRC-1. These coactivators contain one or more "LXXLL boxes" that are responsible for nuclear receptor binding, where L is leucine and X is any amino acid in the sequence motif.

Phytoestrogens
The phytoestrogen, genistein, is completely buried within the hydrophobic core, but H12 does not adopt the distinctive "agonist" position. Instead, H12 lies in a similar orientation to that observed with ER antagonists. The suboptimal alignment of the transactivation helix results in genistein's partial agonist character with ERb. The following is the structure of ERb complexed to genistein and raloxifene:

ERb Ligand-Binding Domain Complexed to Genistein	ERb Ligand-Binding Domain Complexed to Raloxifene

H12 is shown as a magenta coil in the "green sububits". The other subunit in each structure is displayed as "Cartoons" with "Structure" coloring (RasMol). The ligands are Spacefill.

Estrogen: It's Not Just for Females!
Estrogen has been shown to influence the morphology and function of the secretory epithelial cells in rat prostates, making it likely that at least some of the effects of estrogens in vivo are direct rather than indirect. It is unclear whether the described effects are mediated by ERa or ERb, or perhaps even by both receptors, although the expression of ERb mRNA and protein seems to be higher than that of ERa. Another interesting fact is that that plasma estrogen levels increase with age in men. Low levels of estrogen are one of the key factors in bringing on osteoporosis, as estrogen helps maintain bone strength in both men and women. The "Osteoporosis in Males" page, linked below explains this further.

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Selected References

Moras, D & Gronemeyer, H (1998) "The nuclear receptor ligand-binding domain: structure and function". Curr Opin Cell Biol 10:384-391.
**This paper gives a good overview of the details surrounding silencing and transactivation abilities of the estrogen receptor. Their explanations are sequential and easy to follow. It clarifies much of the coactivator and corepressor recruitment that is alluded to in many other papers regarding this topic.

Pettersson, K & Gustafsson, J-Å (2001): "Role of estrogen receptor beta in estrogen action". Ann Rev Physiol 63:165-192.
**Summary of molecular and physiological aspects of ER functions.

Glass, C & Rosenfeld, MG (2000): "The coregulator exchange in transcriptional functions of nuclear receptors". Genes Dev 14:121-141.
**Extensive description of transcriptional coactivators and corepressors in the larger context of the NR superfamily. A good starting point for another Independent Reading project.

Simpson, ER & Davis SD (2000) "Another role highlighted for estrogens in the male: Sexual behavior". PNAS 97:14308-14340.
**Short review and Commentary on a research paper in the same issue of PNAS.

Research Papers
Pike, ACW, et al. (1999) "Structure of the ligand-binding domain of estrogen beta in the presence of a partial agonist and a full antagonist". EMBO J 18:4608-4618, 1999.
**This paper gives good insight into the intricacies of the ER in regards to the minute comformational changes brought upon by binding of raloxifene and genistein, and how these changes propagate to affect transcriptional activity.

Gehm, BD, et al. (1997) "Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor". PNAS 94:14138-14143.
**Figure 1 shown above.

Stauffer, SR, et al. (2000) "Pyrazole ligands: Structure-affinity/activity relationships and etsrogen receptor-a-selective agonists". J Med Chem 43:4934-4947.
**Synthesis and characterization of a compound with ~400-fold binding preference for ERa. Binding measurements, transcription assays, and structural modeling are reported.

Mak, HY, et al. (1999) "Molecular determinants of the estrogen receptor-coactivator interface". Mol Cell Biol 19:3895-3903.
**Mutagenesis experiments show that more than the LXXLL motif is required for specifc binding.

Gee, AC, et al. (1999) "Coactivator proteins have a differential stabilizing effect on the binding of estrogens and antiestrogens with the estrogen receptor". Mol Endocrinol1912-1923.
**A peptide corresponding to the SRC-1 NR Box 2 was used with a fluorescent estrogen analog to measure binding to the protein complexes.

Thornton, JW (2001) "Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial expansions". PNAS 98:5671-5676.
**Phylogenetic analysis used to propose the relationships between all steroid receptors and a possible role for orphan receptors in vertebrate evolution.

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Links to Related ER Sites

NucleaRDB: An Information System for Nuclear Receptors
http://receptors.ucsf.edu/NR/

Nuclear Receptor Resource: Structures, Graphics, etc.
http://nrr.georgetown.edu/NRR/NRR.html

The Steroid Receptor Associated Proteins Resource: at Medical College of Ohio
http://www.mco.edu/depts/pharm/srapr.html
Nuclear Hormone Receptors
http://www.ks.uiuc.edu/Research/pro_DNA/ster_horm_rec/

Osteoporosis in Males
http://uwcme.org/courses/bonephys/opmale.html

Environmental Estrogens and Other Hormones (EEOH)
http://www.tmc.tulane.edu/ecme/eehome/

Chime Pages Elsewhere
Estrogen Receptor (Steroid Binding Domain): Overview and part of a tutorial on several proteins.
http://www.amherst.edu/~pbohara/biochem_30/chime/frameset-h.htm
Estrogen Receptor (DNA Binding Domain)
http://www.amherst.edu/~pbohara/biochem_30/chime/frameset-i.htm

ER LBD: Features of the Tanenbaum, et al. (1998) structure.
http://srv2.lycoming.edu/~newman/courses/bio43598/ER-ligand/index.html
ER DBD
http://srv2.lycoming.edu/~newman/courses/bio43598/ER-DNA/index.html

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Estrogen Receptor Structure Models

The following links are to Chime pages showing models of the ligand-binding domain (LBD) or the DNA-binding domain (DBD) as dimers. In cases where the PDB file was a monomer, the dimer coordinates were obtained from the EMBL Macromolecular Structure Database. Citations to the structure papers (and PDB files) used for these displays are provided on each page. 

A. Estrogen Receptora   ERa complexes with bound:

Estradiol (EST), an agonist.

Raloxifene (RAL), an antagonist.

These two structures were the first reported for an ER LDB. The ligand binding site and different conformations of H12 were described for agonist and antagonist complexes.

Diethylstilbestrol (DES), an agonist & the NR Box II peptide.

Tamoxifen (OHT), an antagonist.

A second agonist-antagonist comparison. This work also reported the structure of the agonist complex with a bound peptide containing the LXXLL motif from a coactivator.

Estradiol (EST), in an unusual tetrameric structure.

The tetrameric form of ER is unlikely to be physiologically relevant, however, the structure does demonstrate the conformational variability of H12.

B. Estrogen Receptorb   ERb complexes with bound:

Genistein (GEN), a partial agonist.

Raloxifene (RAL), an antagonist.

These two structures are very similar to ERa despite only 59% amino acid identity. They also account for the partial agonist and full antagonist features of GEN and RAL.

C. Structural Alignments   Pairwise superimposed comparisons:

The coordinates for each pair of monomers listed were aligned at the Combinatorial Extension Server. The Chime pages provide views of both chains superimposed or of each chain separately. (Bound ligand is included in only one of the models.)

ERa (EST) and ERb (GEN), ERa vs. ERb in agonist complexes.

ERa (RAL) and ERb (RAL), ERa vs. ERb in antagonist complexes.

ERa: DES and OHT, ERa: agonist vs. antagonist complex.

ERa: EST, Wild type ER vs. a triple mutant (Helix 12 in the antagonist conformation).

ERa: EST, The Brzozowski, et al. (1997) vs. Tanenbaum, et al. (1998) models.

"Helix 12 Gallery": ERa-EST vs. All five ER-antagonist (SERM) models.

The various positions of Helix 12 are displayed without the details of the above pairwise comparisons.

D. DNA-Binding Domain:

ER-DBD Complex   Base pair & backbone contacts.

This structure is the only ER protein-DNA complex currently in the PDB. The high sequence identity between the two ER isoforms (97%) suggests that their DNA complex structures would be nearly identical.