Estrogen Receptor
Created by Bilal Chaudhry
Estrogen receptor (2YAT) from Homo sapiens is a nuclear receptor that is used as a binding site for estrogen. The complex it forms plays a vital role in normal sexual and reproductive function. Estrogens physiological role in the body is broad and includes functions such as cardiovascular health, cognition, and behavior. Because of estrogens profound physiological function within the body, it provides major implications in the progression of diseases (9). Since estrogen receptors mediate the effects of estrogen, it is a vital biomarker in determining the response to therapeutic treatments, specifically with selective estrogen modulators (SERM) and selective estrogen receptor down regulators (SERDS) in endocrine therapy (9).
Estrogen induces cellular changes through mechanisms to which this estrogen binds to estrogen receptors (ER). The estrogen can bind to an estrogen receptor through two different mechanisms. The first is the classical mechanism, where the estrogen diffuses into the cell and binds to the ER. This estrogen-ER complex then binds to estrogen response element sequences which results in increased or decreased mRNA levels, associated protein production, and a physiological response. The other mechanism is a nongenomic mechanism in which the estrogen acts more quickly than the classical mechanism. This process results in increased levels of Ca2+/NO and the activation of kinases as this process occurs in the ER in or adjacent to the plasma membrane or through non-ER plasma membrane (7). The molecular weight of estrogen receptor is 28736.36 Da and its isoelectric point (pI) is 6.34, as provided by the Expasy Database (5).
Estrogen receptor has two forms that predominately exist; Estrogen Receptor Alpha (ERα) and Estrogen Receptor Beta (ERβ). The ERα and ERβ differ structurally due to these receptors being encoded by two separate genes, the ESR1 for the ERα and ESR2 for the ERβ. Both of these types of estrogen receptors contain domains that are evolutionarily conserved structurally and functionally (7). The most vital and conserved domain is the DNA-binding domain (DBD) where DNA recognition and binding occurs. The ligand binding domain (LBD) is located on the carboxyl terminal of the estrogen receptor. The component that differentiates the two estrogen receptors is the amino terminal domain, which has varying length and sequence. Both types of estrogen receptors have a high similarity in sequences excluding the amino terminal domain. As a result there is a similar binding affinity for ligands and DNA response elements between ERα and ERβ (7).
ERα is the biomarker for assessment and treatment of endocrine therapy in ER-positive breast cancers. There are drugs being made that have a competitive binding affinity similar to 17β-estradiol (E2). The SERM that is used for treatment of breast cancer is estradiol-pyridine tertra acetate (EPTA-Ln) which has a binding affinity to ERα. Ln here is any lanthanide ion, which for the EEu ligand is the Eu3+ ion. The position of attachment of the EPTA-Ln to the ERα is at the C17 position rather than the C7 position. This allows for EPTA-Ln to exhibit agonist behavior similar to E2, which enhances cell proliferation degradation of ERα in ER-positive breast cancer cells. There have been multiple types of SERMs produced that modify the conformation of the C-terminal helix which allows for varying surface area on the LBD (9).
The crystal structure of the ER-LBD shows that there is a three layer α-helical “sandwich” fold and it has a ligand that is buried within a hydrophobic ligand binding pocket (LBP) on the lower end of the ligand binding domain. The ligand binding pocket of the lower portion of the LDB is more flexible than the upper LBD. The conformation of the C-terminal helix is important in determining cofactor binding and transcriptional activation of ERα. The flexibility of the LBP provides for the expansion in different directions depending on the chemical properties of the bound ligand. This feature is exhibited in the protein-ligand complex of ERα-LBD with EPTA-EU (EEu). The ligand EEu can be described in having “a steroid core with and extended side chain”. This side chain suggests that the ligand is an antagonist. However, because the chirality of carbon 17 of the E2 moiety and the triple bond linkage with the Eu moiety, the orientation of this complex is fixed in the LBP pointing towards Helix 7 (residues 412-417). The H7 region is important because it gets deformed in to an extended loop allowing the LBP to increase its volume by 40% allowing the EEu ligand to bind (9).
The entry and the exit of ligands from the binding domain are dependent on the chemical nature of the ligand and the starting structure, which suggests a clamp model for ligand binding. The binding of the EEu ligand to the ERα-LBP does not alter the dimerization of the protein. The protein exhibits the same dimeric arrangement as when E2 is bound to ERα-LBD. The EEu ligand binds with the ERα complex by having the phenolic hydroxyl group of EEu form hydrogen bonds with the carboxylate of Glu-353, guanidine of Arg-394, carboxyl group of Leu-387 and a free standing water molecule. In addition to this the 17- β hydroxyl group of the EEu forms a hydrogen bond with the residue His-524 (9). There are also some electrostatic forces with Glu-423 and some van der Waal interactions to further stabilize this binding (4). The ligand binding domain has the EEu oriented in a way where the Eu tagged side chain is pointing to the space towards the two-turn helix (residues 412-423) which allows for hydrogen bonding to occur between these residues and the ligand. The hydrogen bonding, which provides most of the free binding energy, along with the hydrophobic effects of the residues stabilizes this ERα-LBD/EEu complex (6). The ERα-LBD/EEu complex is important because it allows for the production of anti-estrogenic derivatives that are specific for targeting ER-positive cells. There is also the formic acid (FMT) ligand which shows up in two instances in ERα. A molecule of formic acid forms hydrogen bonds with His-356 and a free water molecule while another molecule of formic acid forms hydrogen bonds with Leu-327 (4).
This model of the ERα-EEu complex suggests that the clamp that is responsible for locking the ligand in place is composed of three regions which include helices 3, 7-8, and 11. To demonstrate the nature of the clamp, three different ligands were attached to compare the conformation changes of the LBD. When the E2 ligand binds, the clam tightens to form the LBP by having hydrogen bonding occur between the hydroxyl group and residues His-524, Lys-531, Glu-419, and Glu-339 (1). When the EEu ligand binds, there is a Eu tag that disrupts the conformation of helix 7. This shortens the helices 3 and 8 which leads to an open conformation of the clamp. The third ligand of 4-hydroxytamoxifen (OHT) cannot stabilize any of the three helices regions, which results in high flexibility of the LBP. The conformation changes in helices 3 and 11 are most likely due to position of helix 8 (residue 418-423) moving towards these two helices. This suggests that the channel permitting ligands that move in and out of the LBP are due to the cavity formed by the three regions (H3,H7-H8,H11) (9).
ERα consists of only one subunit that has 252 residues. The secondary structure of ERα is 60% helical, which contains 11 helices and is comprised of 165 residues. There are also 2% beta sheets in the secondary structure of ERα consisting of two anti-parallel strands that are comprised of 7 residues (4). The secondary structure of ERα is primarily alpha helices and the architecture of ERα indicated is an orthogonal bundle. There is a two turn alpha helix present from residues 412-423 with the loop being at residues 418-421. There is also a ω loop present in ERα that has a loop between residues 331-340 (9).
Glucocorticoid Receptor (pdb ID=3K23) has approximately 26% sequence similarity to estrogen receptor. The results of DALI (Z=25.6 rmsd=1.8) and Protein Blast (E=9e-28) searches show that glucocorticoid receptor has primary and secondary similarities to estrogen receptor (2) (8). The Dali server compares the tertiary structure and the intermolecular distances for the comparison proteins. The DALI server then comes up with a Z score, where a score above two indicates similar folds. The Z score is the standard deviation away from the mean of the normal population, which in this case means the higher the Z score, the more homology between the proteins (2). The PSI-BLAST looks at the primary structure of a protein to find similar proteins by assigning gaps; an E score below .05 indicates high similarity (8). The lower the E score, the higher the homology between the proteins. Both the estrogen and glucocorticoid receptors both have LBP that have plasticity and flexibility that provide for it to expand in multiple directions depending upon the chemical properties of the bound ligand. However, the estrogen receptor has an N-terminal DNA binding domain while the glucocorticoid receptor has an N-terminal regulatory domain. The DNA binding domain is not located on the N-terminal of the glucocorticoid receptor; whereas, the ligand binding domain is located on the C-terminal domain of the ER. Both of these nuclear receptors have opportunities for the design of unique modulators for the LBD in endocrine therapy (3).