Student Project 10 for UMass Chemistry 423 Spring 2015

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Contents 1 Estrogen receptor beta/genistein complex 2 Introduction 3 Overall Structure 4 Binding Interactions 5 Additional Features 6 Quiz Question 1 7 See Also 8 Credits 9 References

Estrogen receptor beta/genistein complex

by Brian Cain, James Conner, William Doherty, Kyle Missaggia, Anya Novikova, Soo Lim Eunice Park

Student Projects for UMass Chemistry 423 Spring 2015

Introduction

spinqualitylabelsall models 1x7j, Beta estrogen receptor (grey and green) with transcription intermediate factor-2 peptide (pink and yellow) and genistein Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Discovered in 1996, (ER-β) is one of the two isoforms of the estrogen receptor, a ligand-activated transcription factor which regulates the biological effects of the steroid hormone 17 beta-estradiol, or estrogen, in both males and females. The complex is a hetero-tetrameric assembly consisting of 4 molecules and a ligand: 2 copies of , 2 copies of , and the ligand, . Once the ligand is bound, the complex recruits the steroid receptor coactivators, which recruit other proteins to form the transcriptional complex for initiation of transcription^[1]. This activates expression of reporter genes containing estrogen response elements. Genistein is a phytoestrogen with structural similarity to estrogen which competes for estrogen receptors. This ligand can increase growth rate of estrogen receptor expressing breast cancers and can inhibit immune response to cancer cells, allowing them to proliferate depending on its concentration. In normal tissues, ER-β is an essential receptor for maintaining the functions of vital organs, and may also help regulate apoptosis, control antioxidant gene expression, and modulate immune responses^[2].

Although estrogen receptor beta is widely expressed, it is not the primary estrogen receptor in most tissues. As a result, it has become a target for drug delivery, especially since it is 40x more selective for genistein than the α isoform. This enhanced selectivity may be caused by differences in residues ^[3] between the two isoforms, allowing ER-β to accommodate more polar substituents in its binding pocket. ER-β differs greatly from ER-α at the N-terminal domains^[4] , which can be seen located at opposite ends from the C termini in this . The protein is composed of three sections: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain.

N

C

Overall Structure

spinqualitylabelsall models ERβ and genistein complex: β sheet portion (green) and α helices (blue) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The genistein-estrogen receptor beta complex is a heterotetramer consisting of two estrogen receptor beta (ERβ) chains and 2 steroid receptor coactivator-1 chains. Each ERβ contains several domains with specific functions: an N-terminal domain (NTD), a DNA-binding domain (DBD), a flexible hinge region and a C-terminal Ligand-binding domain (LBD). The complex overall is about ^[5].

The is the first activation function (AF-1) domain that consists mostly of random coils and a small portion of helices (red) and sheets (green); it is a . This lack of structure allows the region to recruit and bond many different interaction partners. This region also has the capacity to transactivate transcription without binding estrogen^[6].

The binds estrogen response elements (ERE) of target genes and recruits coactivator proteins responsible for the transcription of these genes. The ERE consist of a palindromic inverted repeat 5'GGTCAnnnTGACC-3' of target genes^[7]. The DBD is a highly . It is composed of two C4-type zinc fingers each containing residues coordinating to a zinc atom^[7].

The hinge region connects the DBD and LBD^[6].

binds estrogen, coregulatory proteins, corepressors and coactivators. Genistein is a plant-derived phytoestrogen not generated by the endocrine system that binds ERβ like estrogen; both ligands are completely buried within the (Hydrophobic, Polar) of the ERβ complex.

Binding at the LBD activates transcription mediated by the DBD. This domain is entirely helical; the LBD interacts with genistein through helices^[6]. The conformationally dynamic portion of this region gives rise to ERβ’s ligand-dependent transcriptional activation (AF-2) function. A key element of AF-2 is helix 12 (H12), which acts as a conformational switch; different receptor ligands influence the orientation of H12. Agonist ligands like genistein position H12 across the ligand-binding pocket of the LBD, which provides a coactivator docking surface. Geinstein binding allows the helices of AF-2 to form a shallow hydrophobic binding site for leucine-rich motifs of coactivators to bind. This conformation provides optimal interaction with coactivators and transcription is activated ^[8].

Binding Interactions

spinqualitylabelsall models pdbcode, Insert caption here Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Genistein's bicyclic form allows it to hydrogen bond on opposite sides with the hydroxyls of the histidine groups on the receptor. binding to the receptor causes a conformational change and activates the receptor resulting in up-regulation for coactivators. Down-regulation will occur in the presence of corepressor as they bind to repressors and indirectly regulate gene expression. In order for the estrogen receptor β genistein to bind to a receptor and activate it there must be stabilization by a coactivator. The coactivator increases the gene expression and with this increase allows it to bind to an activator group consisting of a DNA binding domain. The estrogen receptor is found to be comprised of a dimer attached to a ligand and coactivator peptide which helps to stabilize the structure of each monomer. The conformational state of helix-12 can be modified by the binding of the coactivator ^[9].

This depicts the hydrophobic and hydrophilic residues of the estrogen receptor. The hydrophobic regions are primarily on the inside of the protein surrounding genistein shown in red. Having the hydrophobic residues surrounding the binding pocket will stabilize the structure. The structure of this pocket is tertiary and do to the hydrophobic interactions inside the pocket and hydrophilic interactions on the outside help to stabilize this tertiary structure ^[10]. The is hydrophobic which means that an increase in lipophilicity would increase the affinity for ligands which in this case is genistein. The genistein structure has three hydroxyl groups, an ether and an ester^[11]. These three functional groups are polar and have many possibilities for hydrogen bonding. The His475 and Met336 residues in the binding pocket are capable of forming hydrogen bonds with genistein do to the many hydrogen bond forming functional groups. These residues are different from the residues found in ERα and so the selectivity of genistein is much greater for ERβ ^[12].

Several others factors play a role in ERβ binding, one is steric effects around the binding pocket. The shape of this pocket will create some selectivity based on the size of incoming ligands. Genistein is able to bind because it has the correct size as well as hydrophobic functional groups. If a large bulky ligand tries to bind to the receptor it may have a difficult time do to steric interactions. Another factor is electronic effects such as conjugation which could interfere with binding as well. Genistein is a conjugated system and because it has several oxygen atoms with electron lone pairs electron density could be shifted in a way to help it bind to the ERβ form giving reason to its greater selectivity than ERα.

Additional Features

spinqualitylabelsall models pdbcode, insert caption here Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Since its discovery, the beta strand in the Estrogen receptors have constantly been studied as a potential drug target. This is due to the ERα and ERβ strands being so different that the tissues could potentially be targeted differently with selective ligands for each isoform. What particularly caught the attention of researchers is the fact that ERβ is widely expressed, however it is not the primary estrogen receptor. This could potentially allow drugs to target certain tissues without causing certain estrogenic effects. To validate ERβ’s potential to be a target for drugs is to design highly ERβ selective ligands and then use them as a tool to probe the physiological function of ERβ.

To do this, researchers used an X-ray structure determination of the ERβ ligand binding domain which was complexed with a molecule known as genistein. Using the X-ray structures it was determined that two ideal residue substitutions are ERβ Met336 replacing ERα Leu384 and ERβ Ile373 replacing ERα Met421 ^[13]. These two interactions are capable of having significant contributions to the ERβ selectivity. This favorable selectivity is based on the position and orientation of the side chains relative to the ligand. For example, the side chains allow for an aryl group in the B-ring reign of GEN is capable of making an interaction that is more favorable with ERβ Met 336 instead of ERα Leu384. As for ERβ Ile373 replacing ERα Met421, it is believed that this replacement did not directly affect the selectivity of ERβ, but instead the introduction of the GEN 5-OH sidechain prevents ERα Met421 from interacting as favorably when compared to ERβ Ile421. This means that certain side chains being introduced to the estrogen receptor allows for favorable drug targeting by allowing the ERβ strand become a more favorable binding site for the drug.

Quiz Question 1

spinqualitylabelsall models 1x7j, Beta estrogen receptor (grey and green) with transcription intermediate factor-2 peptide (pink and yellow) and genistein Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

First, we can see the initial view of the complex. Upon visualizing the estrogen receptor in an arrow formation, , the structure can be classified as parallel or anti-parallel. Here is the zoomed . How would would this structure differ in a solution of Hexane?

a. parallel, hydrophobic core would rearrange to outside of the structure because hexane is non-polar

b. parallel, structure would remain the same

c. anti-parallel, hydrophobic core would rearrange to outside of the structure because hexane is non-polar

d. anti-parallel, structure would remain the same

See Also

• UvrABC
• Androgen receptor
• Estrogen receptor
• Estrogen-related receptor
• Nuclear receptor coactivator
• Hormone
• Glycogen synthase kinase 3
• CREB-binding protein

Credits

Introduction - Brian Cain

Overall Structure - Anna Novikova

Drug Binding Site - Kyle Missaggia

Additional Features - William Doherty

Quiz Question 1 - James Conner

References

1. ↑ Grabowski J. Clonidine treatment of clozapine-induced hypersalivation. J Clin Psychopharmacol. 1992 Feb;12(1):69-70. PMID:1552044
2. ↑ Grabowski J. Clonidine treatment of clozapine-induced hypersalivation. J Clin Psychopharmacol. 1992 Feb;12(1):69-70. PMID:1552044
3. ↑ Manas ES, Xu ZB, Unwalla RJ, Somers WS. Understanding the selectivity of genistein for human estrogen receptor-beta using X-ray crystallography and computational methods. Structure. 2004 Dec;12(12):2197-207. PMID:15576033 doi:10.1016/j.str.2004.09.015
4. ↑ Edsall RJ Jr, Harris HA, Manas ES, Mewshaw RE. ERbeta ligands. Part 1: the discovery of ERbeta selective ligands which embrace the 4-hydroxy-biphenyl template. Bioorg Med Chem. 2003 Aug 5;11(16):3457-74. PMID:12878140
5. ↑ Mocklinghoff S, Rose R, Carraz M, Visser A, Ottmann C, Brunsveld L. Synthesis and crystal structure of a phosphorylated estrogen receptor ligand binding domain. Chembiochem. 2010 Nov 2;11(16):2251-4. PMID:20922740 doi:10.1002/cbic.201000532
6. ↑ ^{6.0 6.1 6.2} Raj Kumar, Mikhail N. Zakharov, Shagufta H. Khan, et al., “The Dynamic Structure of the Estrogen Receptor,” Journal of Amino Acids, vol. 2011, Article ID 812540, 2011. DOI:10.4061/2011/812540
7. ↑ ^{7.0 7.1} Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH. CDD: NCBI's conserved domain database. Nucleic Acids Res. 2015 Jan;43(Database issue):D222-6. doi: 10.1093/nar/gku1221. , Epub 2014 Nov 20. PMID:25414356 doi:http://dx.doi.org/10.1093/nar/gku1221
8. ↑ Nina Heldring, Ashley Pike, Sandra Andersson, et al., "Estrogen Receptors: How Do They Signal and What Are Their Targets," Physiological Reviews, Jul 2007, 87 (3) 905-931; DOI: 10.1152/physrev.00026.2006
9. ↑ Manas ES, Xu ZB, Unwalla RJ, Somers WS. Understanding the selectivity of genistein for human estrogen receptor-beta using X-ray crystallography and computational methods. Structure. 2004 Dec;12(12):2197-207. PMID:15576033 doi:10.1016/j.str.2004.09.015
10. ↑ Edsall RJ Jr, Harris HA, Manas ES, Mewshaw RE. ERbeta ligands. Part 1: the discovery of ERbeta selective ligands which embrace the 4-hydroxy-biphenyl template. Bioorg Med Chem. 2003 Aug 5;11(16):3457-74. PMID:12878140
11. ↑ National Center for Biotechnology Information. PubChem Compound Database; CID=5280961, http://pubchem.ncbi.nlm.nih.gov/compound/5280961 (accessed Apr. 5, 2015).
12. ↑ Edsall RJ Jr, Harris HA, Manas ES, Mewshaw RE. ERbeta ligands. Part 1: the discovery of ERbeta selective ligands which embrace the 4-hydroxy-biphenyl template. Bioorg Med Chem. 2003 Aug 5;11(16):3457-74. PMID:12878140
13. ↑ Eric S Manas, Zhang B. Xu, Rayomand J. Unwalla, William S. Somers, “Understanding the Selectivity of Genistein for Human Estrogen Receptor-β Using X-Ray Crystallography and Computational Methods,” Structure, vol. 12, Issue 12, Pages 2095-2277; DOI:10.1016/j.str.2004.09.015