Tamoxifen,
Diethylstilbesterol and the Estrogen Receptor Ligand Binding Region
Sara
Pecorak '04 and Tom Susman '04
Contents:
I.
Introduction
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).
back to top
II.
General Structure of Estrogen/ Estrogen Receptor Complex
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 ligand binding
domain, and is complexed with estradiol. ERa Ligand binding
domain (LBD) contains twelve
a-helices, two
b- sheets, all connected with several short
straight amino acid chains (in white)
. The ERa
LBD has two functionally important regions,
the first being the ligand binding pocket
(estrogen in white)
. 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,
Arg-394, and His-524
(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,
alanine, methionine,
glycine, phenylalanine,
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.
The
coactivator
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). Helix
12, and its high degree of mobility in the
presence of different ligands, illustrates the major functional element
of the estrogen
receptor
(4).
back to top
III.
Structure of the Synthetic Estrogen (DES)/ Estrogen Receptor Complex
(DES) is a synthetic estrogen. The structures
of the two molecules are not the same.
However,
DES can mimic the function of a natural estrogen and act as ligands because
it has similar interactions with the ERa LBD (DES
in red)
. The LBD consists of a hydrophobic cavity formed by residues
from helices 3,6,7,8, 11, and 12 (3). DES
makes three hydrogen bonds with residues of the LBD, His
524, Glu 353,
Arg 394
. 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,
Phe 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. Click
here
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).
back
to top
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 3
and 11, causing
the formation of several new hydrophObic
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 A'
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
and Arg-394,
b as opposed to the 3 made by both DES and estrogen. These binding
differences cause portions of helices 3,
8,
and 11
to extend
(3).
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
Leu-540, Leu-544,
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
of ERa.
back to top
V.
References
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 95:
927-937.
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
Domains. Proceedings of the
national Academy of Sciences of the United States of America 95:
5998-6003.
back to top