Tamoxifen, Diethylstilbesterol and the Estrogen
Receptor Ligand Binding Region
Sara Pecorak '04 and Tom Susman '04
Recent studies have found that high estrogen levels
combined with a genetic predisposition to breast cancer result in a higher
occurance of the disease (3). Estrogen has many
functions throughout the body, many of which are regulated through an activated
estrogen receptor transcription factor. These transcription factors,
in disease prone patients, can activate genes which lead to uncontrolled
cancerous growth. Researchers have hypothesized that altering the
binding of the estrogen to the estrogen receptors can be used in the prevention
and treatment of breast cancer (1).
Estrogen binds to the Estrogen receptor which
then allows the receptor to bind DNA at the estrogen response element (ERE),
a cis-acting enhancer sequence. The estrogen-receptor-ERE complex
then initiates transcription of genes related to the reproductive cycle.
Tamoxifen has been found to be a competitive inhibitor of estrogen-estrogen
receptor a (ERa)
binding (3). This drug has been administered clinically
and found to decrease the occurance of breast cancer in disease prone patients.
Patients undergoing treatment with tamoxifen are found to have decreased
breast tissue density, an indication of a lowered breast cancer risk (1).
Crystal structures of the ERa in complex with
estrogen, a synthetic estrogen, and 4-hydroxytamoxifen provide insights
into the structural changes caused by this competitive inhibition.
Tamoxifen binds to the ligand binding domain of ERa
and causes a conformational shift of helix 12 into an adjacent coactivator
site which in turn prevents ERa from binding
a coactivator (NR box peptide) which would then activate a specific DNA
sequence, the estrogen response element (ERE) (2, 3).
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II. General Structure of Estrogen/ Estrogen Receptor
A common natural estrogen found in human systems is estradiol, commonly
refferred to as E-2. Estradiol
and its analogs are characterized by having a 3-hydroxy group as opposed
to a 3-keto group found in many other steroids (4). These
hydroxyl groups are critical to the overall structre of the receptor complex.
The estrogen receptor featured here is the estrogen receptor a
binding domain, and is complexed
with estradiol. ERa Ligand binding domain
(LBD) contains twelve
all connected with several short straight amino acid chains (in white)
has two functionally important regions, the first being the ligand
binding pocket (estrogen
in green) <>.
The estradiol is oriented in the ligand binding pocket with hydrogen bonding
and van der Waals contacts. Functionally important polar amino acids in
the binding pocket include Glu-353,
(4). Glu-353 interacts in a H-bond with the 3-hydroxy
group. The sidechain of Glu-353 is itself braced by a water mediated H-bond
to Arg-394, which is further braced by a H-bond to the carbonyl of a neighboring
phenalyanine (Phe-404) (4). The Phe-404 is involved in
a hydrophobic interaction to A-ring of the estradiol, thus making the polar
OH group mediated through a hydrophobic interaction (4).
Eighteen different amino acid residues, including leucine,
and isoleucine (in white), located between amino acid number 340 and 530
create hydrophobic bonds to the estradiol <>.
These interactions are esstential for stabilizing the nonpolar elements
of the estradiol ring structure.
site, a necessary binding region for transcriptional activation, is
composed of exposed surfaces from helices 3, 4, 12, and a signature loop.
It should be noted that the extended helix 12 is an anomaly of the crystallization
conditions used to make this chime image (4). The major
stabilizing factor of the coactivator site includes a hydrophobic interaction
between a glutamate in helix 12 and hydrophobic residues in helix 4 . Also,
Lys-362 and Val-364 in the loop between helices 3 and 4 (the signature
sequence) are also essential for coactivator interaction (4).
12, and its high degree of mobility in the
presence of different ligands, illustrates the major functional element
of the estrogen receptor
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III. Structure of the Synthetic Estrogen (DES)/
Estrogen Receptor Complex
is a synthetic estrogen. The structures
the two molecules are not the same but DES can mimic the function of a
natural estrogen and act as ligands because it has similar interactions
with the ERa LBD (DES
The LBD consists of a hydrophobic cavity formed by residues from helices
3,6,7,8, 11, and 12 (3).
makes three hydrogen bonds with residues of the LBD, His
524, Glu 353,
Ring A prime binds to the residues contributed by helices 7, 8, and 9,
mimicking the interactions of the estrogen C
and D rings (3). The protruding ethyl groups
of DES make 4 additional non-polar contacts to Ala
350, Leu 384,
404 and Leu 428
which are not made by estrogen <>
(3). DES also contacts Met
421 and Met 528
which are not made by estrogen <>
(3). The position of helix 12 is also different.
Instead of extending from the protein, it is folded onto the molecule.
to see a comparison of helix 12 posistion in the three different complexes.
When DES binds to the ERa
, a hydrophobic groove is formed by helices
3, 4, 5 and 12 and the turn between helices
3 and 4 <>
(3). This groove is known as the NR
box, the binding site of the NR
Box peptide which is a coactivator.
The NR box peptide contains
a signature motif, LXXLL<>
(X=any residue) (3).
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IV. Structural Changes caused by 4-hydroxytamoxifen
There are two critical differences between the structure of 4-hydroxytamoxifen
(OHT) and estrogen-like substances. It lacks a second hydoxyl
group and has an additional tail extending from the A ring containing an
oxygen and a nitrogen. These differences account for the overall
changes in the ERa when 4-hydroxytamoxifen
is bound to the LBD. In the ligand binding pocket, there are several
hydrophobic interactions that are similar
to those made by DES but there are also significant changes<>.
The dimethylaminoethyl sidechain
which extends from ring C of OHT extends into a space between helices
and 11, causing
the formation of several new hydrophbic interactions
and a cascade effect changing conformations throughout the entire molecule<>
(3). As a result of this sidechain, the B
ring of OHT is forced more deeply into the binding pocket than the
ring of DES causing further structural changes in the ERa
(3). OHT makes makes only 2 hydrogen
bonds with the ligand binding pocket, Glu-353
as opposed to the 3 made by both DES and estrogen. These binding
differences cause portions of helices 3,
and 11 to extend<>
The most significant change in structure caused
by OHT binding is the repositioning of helix 12. Helix
12 repositions over the NR
box coactivator site and prevents binding
of the NR box protein<>.
The conformation of the NR box is unchanged. Helix 12 proteins
and Met-543 replace
the NR box protein LXXLL motif<>
(3). Without this coactivator binding, the ERa
remains unactivated and cannot activate the ERE gene nullifying the function
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1. Atkinson C., R. Warren, S.A.
Bingham, and N.E. Day. Mamographic patterns as a predictive
biomarker of breast cancer risk: effect of tamoxifen.
Cancer Epidemiology, Biomarkers and Prevention 8: 863-866.
2. Klinge, Carolyn M., Sarah
C. Jernigan, Stacy l. Smith, Valentyn V. Tyulmenkov, and Peter C. Kulakosky.
2001. Estrogen response element sequence impacts the conformation
and tranxcriptional activity of estrogen receptor alpha.
Moleculat and Cellular Endocrinology 174: 151-166.
3. Shiau, Andrew K., Dannielle
Barstad, Paula M. Loria, Lin Cheng, Peter J. Kushner, David A. Agard, and
Geoffrey L. Greene. 1998. The Structural Basis of Estrogen Receptor/Coactivator
Recognition and the Antagonism of this Interaction by Tamoxifen. Cell
4. Tanenbaum, David M., Yong
Wang, Shawn P. Williams, and Paul B. Sigler. 1998. Crystallographic
comparison of the Estrogen and Progesterone Receptor's Ligand Binding
of the national Academy of Sciences of the United States of America
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