3D Models of MBP, a Biologically Active Metabolite of Bisphenol A, in Human Estrogen Receptor a and Estrogen Receptor b Michael E. Baker1*, Charlie Chandsawangbhuwana2 1 Department of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Department of Bioengineering, University of California San Diego, La Jolla, California, United States of America Abstract Bisphenol A [BPA] is a widely dispersed environmental chemical that is of much concern because the BPA monomer is a weak transcriptional activator of human estrogen receptor a [ERa] and ERb in cell culture. A BPA metabolite, 4-methyl-2,4bis(4-hydroxyphenyl)pent-1-ene [MBP], has transcriptional activity at nM concentrations, which is 1000-fold lower than the concentration for estrogenic activity of BPA, suggesting that MBP may be an environmental estrogen. To investigate the structural basis for the activity of MBP at nM concentrations and the lower activity of BPA for human ERa and ERb, we constructed 3D models of human ERa and ERb with MBP and BPA for comparison with estradiol in these ERs. These 3D models suggest that MBP, but not BPA, has key contacts with amino acids in human ERa and ERb that are important in binding of estradiol by these receptors. Metabolism of BPA to MBP increases the spacing between two phenolic rings, resulting in contacts between MBP and ERa and ERb that mimic those of estradiol with these ERs. Mutagenesis of residues on these ERs that contact the phenolic hydroxyls will provide a test for our 3D models. Other environmental chemicals containing two appropriately spaced phenolic rings and an aliphatic spacer instead of an estrogenic B and C ring also may bind to ERa or ERb and interfere with normal estrogen physiology. This analysis also may be useful in designing novel chemicals for regulating the actions of human ERa and ERb. Citation: Baker ME, Chandsawangbhuwana C (2012) 3D Models of MBP, a Biologically Active Metabolite of Bisphenol A, in Human Estrogen Receptor a and Estrogen Receptor b. PLoS ONE 7(10): e46078. doi:10.1371/journal.pone.0046078 Editor: Vincent Laudet, Ecole Normale Supérieure de Lyon, France Received April 12, 2012; Accepted August 29, 2012; Published October 4, 2012 Copyright: ß 2012 Baker, Chandsawangbhuwana. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mbaker@ucsd.edu BPA has some structural similarity to estradiol and diethylstilbestrol [Figure 1], and, indeed, BPA binds to human estrogen receptor a [ERa] and ERa and is a transcriptional activator of these ERs [17,18,19,20]. However, BPA’s binding affinity and transcriptional activity for these ERs is over 1000-fold lower than that of E2 [17,18,19,20], which makes it unlikely that nM concentrations of BPA would disrupt estrogen physiology. Nevertheless, in vivo studies indicate that BPA is active at 1 nM to 10 nM [8,10,15,21], which raises the possibility that BPA is metabolized to a more active endocrine disruptor. One such candidate metabolite is 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1ene [MBP] [Figure 1], which has about 1000-fold higher estrogenic activity than BPA [22,23]. To begin to understand the structural basis for the high estrogenic activity of MBP and its higher affinity compared to BPA for human ERa and ERb, we constructed 3D models of MBP and BPA in human ERa and ERb. We find that MBP retains key contacts with human ERa and ERb that are important in activation of these receptors by estradiol. We also find that one phenolic ring of BPA can mimic binding of the A ring of E2 to ERa and ERb, which would account for the binding of BPA to these ERs. However, the second phenolic ring on BPA lacks some key contacts that are found between E2 and both ERs, which may explain the lower estrogenic activity of BPA. In addition to elucidating the Introduction One consequence of our industrial society is the presence of novel environmental chemicals that disrupt normal physiological responses in humans, other vertebrates, as well as invertebrates [1,2]. Many of these chemicals are small hydrophobic molecules that resemble steroids, thyroid hormone, retinoids and other lipophilic hormones and, as a result bind to their receptors in vertebrates [3,4,5,6,7]. Some of these chemicals act like hormones, while others act like anti-hormones. In either case, they disrupt normal endocrine physiology. An endocrine disruptor of much concern is bisphenol A [BPA] because it is widely dispersed in the environment due to the presence of BPA in polycarbonate plastics, which are used in containers for food and water, including baby bottles, as well as the linings of metal cans used for food and beverages [8,9,10]. Leaching of the BPA monomer from these sources into food, milk and the environment exposes humans [11,12,13] and wildlife [2,14] to BPA. A consequence of the widespread use of BPA is that over 90% of the general population is exposed to BPA [9,13,15]. BPA levels range from 0.3 nM to 40 nM in maternal plasma and fetal human serum [8,10,11]. Moreover, due to the lipophilic nature of BPA, it can accumulate in fat [16]. PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Figure 1. Structures of MBP, BPA, E2 and DES. MBP, BPA and DES have a phenolic ring that can mimic the A ring on E2 in binding to ERa and ERb. The spacing between the first and second phenolic hydroxyls on MBP and DES is similar to that between C3 hydroxyl and the 17b-hydroxyl on E2. In contrast, the distance between the two phenolic hydroxyls in BPA is shorter than that in E2. doi:10.1371/journal.pone.0046078.g001 dependent dielectric constant of 2 for 50 iterations. We docked MBP and BPA into this PDB file of human ERb with AutoDock 4 and AutoDockVina [27] with the settings used previously for human ERa. The lowest energy complexes of MBP and BPA in ERa and ERb, as calculated by AutoDock 4 and AutoDock Vina, were refined with the Discover 3 software in Insight II. For this energy minimization step, Discover 3 was used with the CVFF force field and a distant dependent dielectric constant of 2 for 10,000 iterations. During this refinement step, both the amino acids on the ERs and MBP and BPA rearrange their positions so as to lower the Gibbs free energy of the complex. interaction of MBP and BPA with both human ERs, this analysis may be useful in designing novel chemicals for regulating the actions of human ERa and ERb. Methods Human ERa [24] was downloaded from the Protein Data Bank [PDB] as a template for docking of MBP and BPA. ChemDraw 3D was used to create PDB files for MBP and BPA, which were docked to human ERa [PDB:1G50] with AutoDock 4 [25,26] and AutoDock Vina [27]. The grid was centered over the estrogen binding site in human ERa. AutoDock 4 was run using the Lamarckian Genetic Algorithm for 250 trials of 5 million energy evaluations. AutoDock Vina was run with a setting of 20 for exhaustiveness and poses for the 100 lowest energies were collected. The crystal structure of ERb complexed with E2 [PDB:3OLS] [28] was selected for docking MBP and BPA. As was found in other ERb structures in the PDB, 3OLS lacks coordinates for five amino acids corresponding to residues 416– 420. To model the missing amino acids, we used the Homology option in Insight II and the 1G50 structure for human ERa as a template. A PDB file of the complete ERb with E2 was refined with Discover 3 with the CVFF force field and a distant PLOS ONE | www.plosone.org Docking Energy Analysis We used X-Score [29,30] and DSX [DrugScore eXtended] [31] to estimate the relative binding energy of MBP and BPA in the various configurations in ERa and ERb. X-Score uses an empirical scoring function to estimate the affinity of a ligand for a protein. DSX uses a knowledge-based scoring function based on the DrugScore formalism [32] to estimate the affinity of a ligand for a protein. In comparing the score of two ligands for a protein, the ligand with the larger negative score has the higher affinity. 2 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Figure 2. Analysis of two 3D models of MBP in human ERa. A. 3D model of MBP in orientation 1 in human ERa. The first phenolic ring on MBP contacts Glu-353, Arg-394 and Phe-404 on ERa and the second phenolic ring contacts Gly-521, His-524 and Leu-525. Favorable van der Waals contacts have a distance of 4.25 Å or less between MBP and amino acids on ERa. B. 3D model of MBP in orientation 2 in human ERa. The first phenolic ring on MBP contacts Glu-353, Arg-394 and Phe-404 on ERa, and the second phenolic ring contacts Gly-521, His-524 and Leu-525. However, in contrast to Orientation 1, the backbone oxygen on Leu-387 does not contact the phenolic hydroxyl on MBP. Phe-404 and Met-421 do not have van der Waals contacts with the linker between the two phenolic rings on MBP. doi:10.1371/journal.pone.0046078.g002 [Figure 1]. BPA also had two poses for one of the rings in ERa and ERb. We analyzed both poses for MBP and BPA in human ERa and ERb. In our analysis of the 3D models of MBP and BPA in both ERs, we use the term ‘‘first phenolic ring’’ to describe the ring that has contacts with ERa and ERb that are similar to the A ring of E2. Results Docking of MBP and BPA to Human ERa and ERb Docking of MBP into human ERa and ERb using AutoDock 4 [25,26] and AutoDock Vina [27] gave two symmetric poses, which is not surprising because MBP has a phenolic ring at each end PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Table 1. Distances between MBP and ERa. Figure 2A ERa MBP Distance Orientation 1 Oe2, Glu-353 O4’ 3.1 Å Ng2, Arg-394 O4’ 3.1 Å O, Leu-387 O4’ 2.8 Å Cb, Leu-391 O4’ 3.7 Å Ce2, Phe-404 C2’ 3.9 Å Ce2, Phe-404 C1 3.8 Å Nd1, His-524 O4’’ 3.0 Å O, Gly-521 O4’’ 2.8 Å Cb, Leu-525 C4’’ 4.0 Å Sd, Met-343 C3’’ 3.7 Å Sd, Met-421 O4’’ 4.1 Å Ce, Met-421 C3’’ 3.8 Å Cd2, Leu384 C6 3.9 Å Cd1, Leu384 C6 3.7 Å Cd2, Thr-347 C5 3.6 Å Cb, Ala-350 C6 3.8 Å Figure 2B ERa MBP Distance Orientation 2 Oe2, Glu-353 O4’’ 2.6 Å Ng2, Arg-394 O4’’ 3.2 Å O, Leu-387 O4’’ 6.6 Å Cb, Leu-391 O4’’ 3.7 Å Cd2, Phe-404 C3’’ 3.9 Å Ce2, Phe-404 C6 4.5 Å Nd1, His-524 O4’ 3.0 Å O, Gly-521 O4’ 2.8 Å Cb, Leu-525 C3’ 3.9 Å Sd, Met-343 C5’ 4.0 Å Sd, Met-421 C5’ 3.9 Å Ce, Met-421 C5’ 3.9 Å Ce, Met-421 C6 5.2 Å Cd1, Leu384 C2’ 3.7 Å Cd2, Leu384 C3’ 3.9 Å Cd2, Thr-347 C1 3.6 Å Cb, Ala-350 C5 3.8 Å doi:10.1371/journal.pone.0046078.t001 ring. The distances between MBP and ERa are shown in Figure 2A and Table 1. For comparison, in Figure 3A and Table 2 we show the distances between E2 and human ERa [33,35,36,37,38]. The first phenolic ring on MBP has contacts that are similar to that of the A ring on E2 with human ERa [33,34,39]. The phenolic hydroxyl on MBP is 3.1 Å from Oe2 on Glu-353, 3.1 Å from Ng2 on Arg-394 and 2.8 Å from the backbone oxygen of Leu-387. MBP is 3.9 Å from Ce2 on Phe-404 [Figure 2A, Table 1]. These contacts are similar to that for E2 with human ERa, except that Cd2 on Leu-387 does not contact the phenolic hydroxyl on MBP, in contrast to the contact between Leu-387 and E2 in human ERa [Figure 3A, Table 2]. Analysis of the crystal structures of ERa complexed with E2 [33,34] and other estrogens [35] revealed that Glu-353 and Arg394 have important stabilizing contacts with the C3 hydroxyl on the A ring and His-524 with the 17b-hydroxyl on the D ring. Glu-305, Arg-346 and His-475 on ERb have similar stabilizing contacts with estrogens. As reported below, the presence or absence of these contacts in the 3D models of ERa and ERb with BPA and MBP is important analyzing the interaction between these chemicals and the ERs. Analysis of MBP in Orientation 1 in Human ERa In Figure 2A, we show the 3D model of MBP in human ERa in Orientation 1, in which C1 on MBP is closest to first phenolic PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Figure 3. Interaction of E2 with amino acids in human ERa and ERb. A. Interaction of E2 with human ERa [24,33,34,36,37,38,39]. The phenolic hydroxyl of E2 contacts Glu-353, Arg-394 and Leu-387. The 17b-hydroxyl contacts His524 and Leu-525. The D ring contacts Met343, Met421, Gly-521 and Ile-424. Favorable van der Waals contacts have a distance of 4.25 Å or less between E2 and amino acids on ERa. B. Interaction of E2 with human ERb [28]. The phenolic hydroxyl of E2 contacts Glu-305, Arg-346 and Leu-339. The 17b-hydroxyl contacts Gly-472, His473 and Leu-476. The D ring contacts Met-336 and Ile-373. Favorable van der Waals contacts have a distance of 4.25 Å or less between E2 and amino acids on ERb. doi:10.1371/journal.pone.0046078.g003 The second phenolic hydroxyl in MBP is 3 Å, 2.8 Å and 4 Å from Nd1 on His-524, the backbone oxygen on Gly-521 and Cb on Leu-525, respectively, on ERa [Figure 2A]. This phenolic hydroxyl also contacts Met-343 and Met-421 on ERa. These PLOS ONE | www.plosone.org five residues stabilize the D ring on E2 in human ERa [Figure 3A]. There are, however, differences in some interactions between ERa and MBP compared to that with E2. While Gly-521 and 5 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Table 2. Distances between E2 and ERa and ERb. Figure 3A ERa E2 Distance Crystal Structure Oe2, Glu-353 O3 2.8 Å PDB: 1G50 Ng2, Arg-394 O3 2.9 Å O, Leu-387 O3 3.0 Å 3.0 Å Cd2, Leu-387 O3 Ce2, Phe-404 C10 3.6 Å Nd1, His-524 O17 2.8 Å O, Gly-521 O17 O, Gly-521 C16 3.4 Å 3.5 Å Cd1 Leu-525 O17 Cd1 Leu-525 C18 3.9 Å Sd, Met-343 O17 3.8 Å Ce, Met-421 O16 3.6 Å Sd, Met-421 O17 Cd2, Leu384 C18 5.2 Å Cd1, Leu384 C18 4.6 Å Cb, Ala-350 C1 4.2 Å Cb, Leu-391 C4 3.8 Å Cd2, Leu-391 C4’ 3.9 Å Figure 3B ERb E2 Distance Crystal Structure Oe2, Glu-305 O3 2.6 Å PDB: 3OLS Ng2, Arg-346 O3 3.0 Å O, Leu-339 O3 3.4 Å Cb, Leu-339 C2 4.0 Å Cd1, Leu-343 C4 4.0 Å 3.9 Å Cd2, Leu-343 C4 Ce2, Phe-356 C5 3.7 Å Cb, Ala-302 C1 3.9 Å O, Gly-472 O17 3.9 Å Nd1, His-475 O17 3.0 Å Cb, Leu-476 O17 3.4 Å Cd2 Leu-476 C18 3.9 Å Ce, Met-295 O17 3.5 Å Sd, Met-336 C18 3.7 Å Ce2, Met-336 C18 3.4 Å Cd1, Ile-373 C16 3.8 Å Ce, Met-421 O17 3.5 Å doi:10.1371/journal.pone.0046078.t002 der Waals contacts with C1 on MBP, which has no equivalent in E2 in ERa. Met-421 contact the second phenolic hydroxyl on MBP [Figure 2A], Gly-521 and Met-421 contact C16 on E2 in ERa [Figure 3A]. While Leu-384 has two van der Waals contacts with MBP, Leu-384 does not contact E2 in ERa. While Thr-347 has a van der Waals contact with MBP, Thr347 does not contact E2 in ERa. While Cb on Leu-391 is 3.7 Å from the first phenolic hydroxyl on MBP, this contact is absent between ERa and E2. While Ala-350 contacts the linker between the two phenolic rings on MBP, Ala-350 contacts C1 on the A ring in E2 in ERa. Phe-404 and Met-421 have van PLOS ONE | www.plosone.org Analysis of MBP in Orientation 2 in Human ERa As shown in Figure 2B and Table 1, analysis of ERa with MBP in Orientation 2 reveals that MBP has contacts with Glu-353, Arg394, Phe-404, Met-343, Leu-384, Met-421, Gly-521, His-524 and Leu-525 that are similar to those found in Orientation 1 of MBP in ERa. Due to the reversed orientation of MBP in ERa, C1 on MBP has a van der Waals contact with Thr-347, and the other part of the linker contacts Ala-350. 6 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Figure 4. Analysis of two 3D models of MBP in human ERb. A. 3D model of MBP in orientation 1 in human ERb. The first phenolic ring on MBP contacts Glu-305, Arg-346, Leu-339, Leu-343 and Phe-356. The second phenolic ring contacts Gly-472, His-475 and Leu-476, which are important in the interaction of the D ring of E2 with ERb. B. 3D model of MBP in orientation 2 in human ERb. The first phenolic ring on MBP contacts the backbone oxygen on Leu-339, Cb on Ala-302 and Leu-343. These contacts are absent between MBP in Orientation 2 in ERa [Figure 2B]. doi:10.1371/journal.pone.0046078.g004 human ERa [Figure 2A, Table 2] and between E2 and ERb [Figure 3B]. Like the A ring in E2, the first phenolic ring on MBP has stabilizing contacts with Glu-305, Arg-346, Phe-356, Leu-339 and Leu-343 in ERb. The second phenolic ring contacts His-475, Gly-472, Leu-476, Ile-373 and Ile-376 [Figure 4A, Table 3]. Analysis of MBP in Orientation 1 in Human ERb Figure 4A shows MBP in Orientation 1 in human ERb. For comparison, in Figure 3B, we show E2 in human ERb [28]. Many of the contacts between MBP and human ERb shown in Figure 4A and Table 3 are similar to that between MBP in Orientation 1 and PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Table 3. Distances between MBP and ERb. Figure 4A ERb MBP Distance Orientation 1 Oe2, Glu-305 O4’ 2.6 Å Ng2, Arg-346 O4’ 3.1 Å O, Leu-339 O4’ 3.4 Å Cb, Leu-343 O4’ 3.9 Å Cd2, Phe-356 C2’ 4.0 Å Ce2, Phe-356 C1 4.0 Å Nd1, His-475 O4’’ 3.0 Å O, Gly-472 O4’’ 3.6 Å Cb, Leu-476 C3’’ 4.1 Å Ce, Met-295 O4’’ 5.7 Å 3.9 Å Cb, Ala-302 C6 Sd, Met-336 C6 3.5 Å Cd1, Ile-373 O4’’ 3.6 Å Cd1, Ile-376 O4’’ 3.2 Å Figure 4B ERb MBP Distance Orientation 2 Oe2, Glu-305 O4’’ 2.6 Å Ng2, Arg-346 O4’’ 3.3 Å O, Leu-339 C4’’ 3.6 Å Cb, Leu-343 C5’’ 4.1 Å Cd2, Leu-343 C6’’ 4.1 Å Cd2, Phe-356 C5’’ 3.9 Å Ce2, Phe-356 C1 4.6 Å Nd1, His-475 O4’ 3.2 Å O, Gly-472 C5’ 3.6 Å Cb, Leu-476 O4’ 3.3 Å Ce, Met-295 O4’ 3.3 Å Cb, Ala-302 C5 4.0 Å Cb, Ala-302 C2’’ 4.0 Å Sd, Met-336 C6 3.5 Å Ce, Met-336 C5’ 4.0 Å Cd1, Ile-373 C1 3.9 Å Cc, Ile-376 C1 3.7 Å Cd1, Ile-376 O4’ 6.0 Å doi:10.1371/journal.pone.0046078.t003 Figure 4B shows MBP in Orientation 2 in human ERb. Many of the contacts between MBP in Orientation 2 and human ERb [Table 3] are similar to that between MBP in Orientation 1 and human ERb [Figure 4A, Table 3]] and between E2 and ERb [Table 2]. The backbone oxygen on Leu-339, Cb on Ala-302 and the side chains on Leu-343 contact the first phenolic ring on MBP. These contacts are absent between MBP in Orientation 2 in ERa [Figure 2B, Table 1]. on Phe-404 and Cb on Ala-350 [Figure 5A, Table 4]. However, the backbone oxygen on Leu-387 is 6.4 Å from the phenolic hydroxyl and Leu-391 does not have a van der Waals contact with the phenolic ring. The second phenolic ring does not contact either Gly-521, His524 or Leu-525 [Table 4]. Instead, phenolic ring moves so that it contacts Ce2 and Cg on Phe-404, Ce on Met-421 and Cd2 on Ile424. Also, Ala-350 and Leu-384 and Thr-347 contact the linker on BPA. Analysis of BPA in Orientation 1 in Human ERa Analysis of BPA in Orientation 2 in Human ERa Figure 5A shows BPA in Orientation 1 in human ERa. The phenolic ring on BPA, corresponding to the A ring of E2, has stabilizing contacts with Oe2 on Glu-353, Ng2 on Arg-394, Cd2 In Figure 5B, we show BPA in Orientation 2 in human ERa. The first phenolic ring on BPA contacts Oe2 on Glu-353, Ng2 on Arg-394, Cd2 on Phe-404 and Cb on Ala-350 [Figure 5B, Analysis of MBP in Orientation 2 in Human ERb PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Figure 5. Analysis of two 3D models of BPA in human ERa. A. 3D model of BPA in orientation 1 in human ERa. The first phenolic ring on BPA contacts Glu-353, Arg-394 and Phe-404 on ERa, but does not contact either Leu-387 or Leu-391. Moreover, the second phenolic ring does not contact either Gly-521, His-524 or Leu-525. Instead, the second phenolic ring contacts Phe-404, Met-421 and Ile-424. B. 3D model of BPA in orientation 2 in human ERa. The first phenolic ring on BPA contacts Glu-353 and Arg-394 on ERa, but does not contact Leu-387 or Phe-404. The second phenolic ring does not contact either Gly-521 or His-524. Instead, the second phenolic ring has novel contacts with Thr-347 and Leu-384. doi:10.1371/journal.pone.0046078.g005 PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Table 4. Distances between BPA and ERa. Figure 5A ERa BPA Distance Orientation 1 Oe2, Glu-353 O4’ 2.6 Å Ng2, Arg-394 O4’ 3.3 Å O, Leu-387 O4’ 6.4 Å Cd2, Phe-404 C3’ 3.9 Å Ce2, Phe-404 C6’’ 3.6 Å Cg, Phe-404 C5’’ 3.6 Å Nd1, His-524 O4’’ 6.8 Å Cd1, Leu-384 C1 3.5 Å Cb, Ala-350 C1 4.1 Å Cb, Ala-350 C1’ 4.0 Å Ce, Met-421 O4’’ 3.3 Å Cd2, Ile-424 O4’’ 3.5 Å Cd2, Thr-347 C3 4.3 Å Figure 5B ERa BPA Distance Orientation 2 Oe2, Glu-353 O4’ 2.6 Å Ng2, Arg-394 O4’ 3.2 Å O, Leu-387 O4’ 6.2 Å Cd2 Leu-391 C6’ 3.9 Å Cd2 Leu-391 C3 4.0 Å Ce2, Phe-404 C3 4.1 Å Nd1, His-524 O4’’ 7.8 Å O, Gly-521 O4’’ 7.4 Å Cb, Leu-525 O4’’ 3.3 Å Cd1, Leu-525 O4’’ 3.5 Å Sd, Met-343 C5’’ 5.7 Å Sd, Met-421 C5’’ 7.7 Å Ce, Met-421 C5’’ 7.6 Å Ce, Met-421 C6’’ 7.1 Å Cd1, Leu384 C3’’ 4.0 Å Cd2, Thr-347 O4’’ 3.4 Å Cb, Ala-350 C2’’ 3.8 Å Cb, Ala-350 C2’ 3.7 Å doi:10.1371/journal.pone.0046078.t004 The second phenolic ring contacts Gly-472, His-475, Leu-476, Ile-373 and Met-336, but the second phenolic ring does not contact Met-295. Table 4]. Leu-391 has a van der Waals contact with the phenolic ring. However, the backbone oxygen on Leu-387 does not contact BPA. The second phenolic ring on BPA does not contact either Gly521, His-524, Met-421 or Ile-424 [Table 4]. Instead, the phenolic hydroxyl contacts Leu-525 and Thr-347. Leu-384 and Ala-350 also contact the second phenolic ring. Analysis of BPA in Orientation 2 in Human ERb In Figure 6B, we show the minimized structure of BPA in Orientation 2 in human ERb. The first phenolic ring on BPA contacts Oe2 on Glu-305, Ng2 on Arg-346, Ce2 on Phe-404, Cb on Ala-350, Cd2 on Leu-343 and the backbone oxygen on Leu339 [Figure 6B, Table 5]. The second phenolic ring does not contact either Gly-472, His475, Leu-476 or Met-295 [Table 5]. Instead, the phenolic hydroxyl contacts Phe-377 and Ile-373. Interestingly, Phe-356 contacts the second phenolic ring and Met-336 contacts the linker on BPA. Analysis of BPA in Orientation 1 in Human ERb In Figure 6A, we show BPA in Orientation 1 in human ERb. The first phenolic ring on BPA contacts Oe2 on Glu-305, Ng2 on Arg-346, Ce2 on Phe-404, Cb on Leu339 and the backbone nitrogen on Ala-302 [Figure 6A, Table 5]. The backbone oxygen on Leu-339 does not contact the phenolic hydroxyl. Cb on Ala302 and Cd2 on Leu-343 do not contact the phenolic ring. PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Figure 6. Analysis of two 3D models of BPA in human ERb. A. 3D model of BPA in orientation 1 in human ERb. The first phenolic ring on BPA contacts Glu-305, Arg-346, and Phe-356, but does not contact either the backbone oxygen on Leu-339 or Cd2 on Leu-343. The second phenolic ring contacts Gly-472, His-475 and Leu-476. B. 3D model of BPA in orientation 2 in human ERb. The first phenolic ring on BPA contacts Glu-305, Arg-346, Phe-356, the backbone oxygen on Leu-339 and Cd2 on Leu-343. The second phenolic ring does not contact either Gly-472, His-475 or Leu-476. doi:10.1371/journal.pone.0046078.g006 PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Table 5. Distances between BPA and ERb. Figure 6A ERb BPA Distance Orientation 1 Oe2, Glu-305 O4’ 2.6 Å Ng2, Arg-346 O4’ 4.3 Å O, Leu-339 O4’ 5.7 Å Cb, Leu-339 O4’ 4.0 Å Cd2, Leu-343 C4’ 5.3 Å Cd2, Phe-356 C3’ 4.0 Å Ce2, Phe-356 C2’ 3.8 Å Ce2, Phe-356 C3 3.8 Å Nd1, His-475 O4’’ 3.2 Å O, Gly-472 O4’’ 2.8 Å Cb, Leu-476 O4’’ 3.5 Å Ce, Met-295 O4’’ 4.3 Å Cb, Ala-302 C6’ 5.5 Å N, Ala-302 O4’ 3.4 Å Sd, Met-336 C2’’ 3.5 Å Ce, Met-336 C3’’ 3.6 Å Cd1, Ile-373 C5’’ 3.7 Å Cd1, Ile-376 C6’’ 3.6 Å Figure 6B ERb BPA Distance Orientation 2 Oe2, Glu-305 O4’ 2.6 Å Ng2, Arg-346 O4’ 3.2 Å O, Leu-339 C4’ 4.0 Å Cd2, Leu-343 C5’ 3.9 Å Ce2, Phe-356 C6’ 4.2 Å Ce2, Phe-356 C6’’ 3.6 Å Cf2, Phe-356 C5’’ 3.7 Å Nd1, His-475 O4’’ 7.1 Å O, Gly-472 O4’’ 8.4 Å Cb, Leu-476 O4’’ 10.2 Å Ce, Met-295 O4’’ 8.5 Å 4.1 Å Cb, Ala-302 C2’ Sd, Met-336 C3 3.2 Å Ce, Met-336 C3 3.7 Å Cd1, Ile-373 C1 3.9 Å Cc1, Ile-376 O4’’ 3.5 Å O, Ile-376 O4’’ 3.8 Å Ce1, Phe-377 O4’’ 3.6 Å N, Phe-377 O4’’ 3.4 Å doi:10.1371/journal.pone.0046078.t005 Docking Energy Analysis Discussion We used X-Score [31] and DSX [31] to estimate the affinity of MBP and BPA in their different orientations in ERa and ERb. Tables 6 and 7 summarize these analyses for the X-Score and DSX. For both algorithms, MBP has an affinity for ERa and ERb that is closer to that of E2 than is BPA for these receptors. This is consistent with previous assays of the activity of MBP and BPA [22,23]. PLOS ONE | www.plosone.org The leaching of BPA monomers from polycarbonate containers and from liners of metal containers for food and beverages has contributed to the widespread exposure of humans to BPA [2,11,12,13,15]. The relatively low affinity of BPA, compared to E2, for human ERa and ERb [17,19,20] would, at first glance, make it unlikely that BPA would be a problem as an estrogenic endocrine disruptor at nM concentrations [9,10,11]. However, it is 12 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb synthetic bisphenols [39,40] indicate the length of the carbon linker between bisphenols and side chain substitutents on the cyclohexane ring on cyclofenils are important in establishing contacts that lead to high affinity binding to the ER. This is consistent with the analysis of our 3D models of BPA and MBP in ERa and ERb as discussed below. Table 6. Docking analysis of MBP and BPA in ERa and ERb. Receptor Ligand Score Figure ERa E2 7.4 Figure 3A ERa MBP Orientation 1 7.2 Figure 2A ERa MBP Orientation 2 7.2 Figure 2B ERa BPA Orientation 1 6.5 Figure 4A ERa BPA Orientation 2 6.5 Figure 4B ERb E2 7.5 Figure 3A ERb MBP Orientation 1 7.1 Figure 5A ERb MBP Orientation 2 7.2 Figure 5B ERb BPA Orientation 1 6.7 Figure 6A ERb BPA Orientation 2 6.7 Figure 6B MBP Retains Important Contacts found between E2 and ERa and ERb Our 3D models of MBP and BPA in human ERa and ERb [Figures 2, 4–6] identify contacts that can explain MBP’s high affinity and BPA’s low affinity for both estrogen receptors. A key structural difference between BPA and MBP is the longer spacing between the two phenolic rings in MBP [Figure 1]. As a result, both phenolic rings on MBP form stabilizing contacts with ERa and ERb that are similar to that between the A and D rings of E2 and human ERa and ERb [28,33,34,35,38,39,41]. These 3D models predict that the second phenolic hydroxyl on MBP has a hydrogen bond with His-524 on ERa and His-475 on ERb. Our 3D models can be tested by investigating transcriptional activation by MBP of ERa and ERb in which His-524 and His-475, respectively, have been mutated. X-Score Analysis of MBP and BPA in ERa and ERb [29,30]. doi:10.1371/journal.pone.0046078.t006 clear that nM concentrations of BPA have estrogenic activity [8,21]. The discovery that MBP, a metabolite of BPA, has a nM affinity for human ERa and ERb, suggests that metabolism of BPA to MBP could explain some of effects of BPA on estrogen physiology [22,23]. There is a structural basis for considering BPA and MBP as potential ligands for ERa and ERb because BPA and MBP have some structural similarities to known synthetic estrogens [Figure 7]. BPA is a bisphenol linked by one carbon atom [Figure 1] as are cyclofenil-type estrogens [Figure 7], some of which have high affinity for ERa and ERb [40]. MBP is a bisphenol linked by three carbon atoms [Figure 1] as is benzestrol [Figure 7], which has a high affinity for ERa and ERb [39]. Hexestrol, which is linked by two carbon atoms, also has a high affinity for ERa and ERb [18,39]. Thus, it is reasonable to be concerned about potential endocrine disruption by synthetic bisphenols. However, as discussed below, our 3D model of BPA in ERs indicates that BPA does not have the contacts with an ER as is found between fluorine-substituted cyclofenil derivatives [40]. Studies with a wide variety of BPA Lacks some Contacts found between E2 and ERa and ERb Like the A ring on E2, one phenolic ring on BPA has stabilizing contacts with Glu-353, Arg-394 and Phe-404 in ERa [Figure 5]. Interestingly, Phe-404 also contacts the second phenolic ring. However, the second phenolic ring on BPA does not contact either Gly-521 or His-524 on ERa, which is significant because contacts between E2 and Gly-521 and His524 in ERa are important in binding of E2 [28,33,35,39,41]. Also, Leu-387 does not contact the first phenolic ring of BPA in either Orientation 1 or Orientation 2. The loss of these contacts between BPA and ERa may explain the lower affinity of BPA for ERa. In the 3D model of BPA in Orientation 1 in ERb [Figure 6A], one phenolic ring on BPA has stabilizing contacts with Glu305, Arg-346 and Phe-356 in ERb. Moreover, the second phenolic ring on BPA contacts Gly-472, His-475 and Leu-476 on ERb. Thus, some of the key interactions between the ERb and the A and D rings on E2 [Figure 3B] are conserved for BPA in Orientation 1 in ERb. However, neither Leu-339 nor Leu-343 contacts the first phenolic ring on BPA. The loss of these contacts would be expected to lower affinity of BPA for ERb. Although BPA in Orientation 2 in ERb [Figure 6B] has contacts with Glu-305, Arg-346, Phe-356, Leu-339 and Leu-343, BPA does not contact either Gly-472, His-475 or Leu-476 on ERb. Instead, the second phenolic ring has novel contacts with Ile-373, Ile-376, Phe-377 and Phe-356. Table 7. Docking analysis of MBP and BPA in ERa and ERb. Receptor Ligand Score Figure ERa E2 2116 Figure 3A ERa MBP Orientation 1 2103 Figure 2A ERa MBP Orientation 2 2110 Figure 2B ERa BPA Orientation 1 284 Figure 4A ERa BPA Orientation 2 291 Figure 4B ERb E2 2123 Figure 3B ERb MBP Orientation 1 2112 Figure 5A ERb MBP Orientation 2 2116 Figure 5B ERb BPA Orientation 1 290 Figure 6A ERb BPA Orientation 2 289 Figure 6B Cellular Context Influences Estrogenic Activity of MBP and BPA The presence of two phenolic rings in MBP and BPA and the flexibility in the estrogen binding site in ERa and ERb [7,33,35,42,43,44,45,46] are important factors in the binding of MBP and BPA to these ERs. The equilibrium dissociation constant of BPA for ERa and ERb is about 195 nM and 35 nM respectively [18]. Using a different binding assay, IC50s [23] for binding of BPA and MBP to ERa and ERb were reported. BPA and MBP have IC50s of 1.8 mM and 52 nM, respectively DSX Analysis of MBP and BPA in ERa and ERb [31,32]. doi:10.1371/journal.pone.0046078.t007 PLOS ONE | www.plosone.org 13 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb Also important for transcriptional activation of ERs and other nuclear receptors by steroids and endocrine disruptors is the binding of co-regulators to the ligand-receptor complex [47,48,49,50,51,52,53]. Thus, even a low affinity ligand such as 27-hydroxy-cholesterol can have transcriptional activity for the ER in the presence of the appropriate co-activator [44]. The interaction of complexes of ERa and ERb with different co-regulators may explain the report by Yoshihara et al. [23] that transcriptional activation of ERa and ERb by MBP and BPA depended on the cellular context. That is, the estrogenic activity of MBP and BPA is altered in the presence or absence of co-regulators [20,48,50,51,54,55]. In the yeast estrogen screening (YES) assay, the EC50 potencies for transcriptional activation of ERa by MBP and BPA were 0.7 mM and 160 mM, respectively. In experiments, which included the Transcriptional Intermediary Factor 2 [TIF2] co-activator in the assay, the EC50s for transcriptional activation of rat ERa with TIF2 by MBP and BPA were 8.3 nM and 14 mM, respectively, and the EC50s for rat ERb with TIF2, by MBP and BPA were 8.3 nM and 13 m M, respectively. Thus, transcriptional activation of ERs by MBP and BPA in an assay containing TIF2, which mimicked conditions in some mammalian cells, increased by about 10fold compared to the assay in yeast cells. Further evidence for the importance of cellular context on transcriptional potency of MBP and BPA comes from experiments using an ERE-luciferase reporter assay in 3T3 cells. Yoshihara et al. [23] found that the EC50s for MBP and BPA for ERa were 0.68 nM and 1 m M, respectively. For ERb in 3T3 cells, the EC50s for MBP and BPA were 0.46 nM and 89 nM, respectively. Together these experiments by Yoshihara et al. suggest that MBP is a potential disruptor of physiological responses that are mediated by ERa and ERb. Although MBP and benzestrol have a three carbon linker between their two phenols, their linkers are different. Despite this difference, nM concentrations of MBP activate transcription of the ER in mammalian cells. This raises the possibility that other environmental chemicals with two phenolic rings connected with novel aliphatic linkers may have a physiologically relevant activity towards ERa and ERb in cells with coactivators that can activate the chemical-ER complex. We also note that the 3D models of MBP in ERa and ERb may be useful in the development of new chemicals for use as selective ER agonists or antagonists. Figure 7. Structures of bisphenols that are potent synthetic estrogens. Bisphenols, linked with one, two or three carbons, can have high affinity for ERs. 3-(3-fluoropropyl)cyclofenil, hexestrol and benzestrol have a higher affinity for ERa that does E2 [18,39,40]. doi:10.1371/journal.pone.0046078.g007 Author Contributions Conceived and designed the experiments: MEB. Performed the experiments: CC. Analyzed the data: CC MEB. Wrote the paper: MEB. for ERa and IC50s of 0.74 mM and 0.12 mM, respectively, for ERb. References 5. le Maire A, Bourguet W, Balaguer P (2010) A structural view of nuclear hormone receptor: endocrine disruptor interactions. Cell Mol Life Sci 67: 1219– 1237. 6. Sladek FM (2011) What are nuclear receptor ligands? Mol Cell Endocrinol 334: 3–13. 7. Baker ME (2011) Insights from the structure of estrogen receptor into the evolution of estrogens: Implications for endocrine disruption. Biochem Pharmacol 82: 1–8. 8. Welshons WV, Nagel SC, vom Saal FS (2006) Large effects from small exposures. III. Endocrine mechanisms mediating effects of bisphenol A at levels of human exposure. Endocrinology 147: S56–69. 1. Colborn T, vom Saal FS, Soto AM (1993) Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ Health Perspect 101: 378– 384. 2. Diamanti-Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, et al. (2009) Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr Rev 30: 293–342. 3. Baker ME (2005) Xenobiotics and the evolution of multicellular animals: emergence and diversification of ligand-activated transcription factors. Integrative and Comparative Biology 45: 172–178. 4. Markov GV, Tavares R, Dauphin-Villemant C, Demeneix BA, Baker ME, et al. (2009) Independent elaboration of steroid hormone signaling pathways in metazoans. Proc Natl Acad Sci U S A 106: 11913–11918. PLOS ONE | www.plosone.org 14 October 2012 | Volume 7 | Issue 10 | e46078 3D Models of MBP in Human ERa and ERb 9. Melzer D, Rice NE, Lewis C, Henley WE, Galloway TS (2010) Association of urinary bisphenol a concentration with heart disease: evidence from NHANES 2003/06. PLoS One 5: e8673. 10. Vandenberg LN, Chahoud I, Heindel JJ, Padmanabhan V, Paumgartten FJ, et al. (2010) Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ Health Perspect 118: 1055–1070. 11. Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M, et al. (2002) Parent bisphenol A accumulation in the human maternal-fetal-placental unit. Environ Health Perspect 110: A703–707. 12. Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV (2007) Human exposure to bisphenol A (BPA). Reprod Toxicol 24: 139–177. 13. Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL (2008) Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003–2004. Environ Health Perspect 116: 39–44. 14. Oehlmann J, Schulte-Oehlmann U, Kloas W, Jagnytsch O, Lutz I, et al. (2009) A critical analysis of the biological impacts of plasticizers on wildlife. Philos Trans R Soc Lond B Biol Sci 364: 2047–2062. 15. Vandenberg LN, Maffini MV, Sonnenschein C, Rubin BS, Soto AM (2009) Bisphenol-A and the great divide: a review of controversies in the field of endocrine disruption. Endocr Rev 30: 75–95. 16. Fernandez MF, Arrebola JP, Taoufiki J, Navalon A, Ballesteros O, et al. (2007) Bisphenol-A and chlorinated derivatives in adipose tissue of women. Reprod Toxicol 24: 259–264. 17. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D (1993) Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132: 2279–2286. 18. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, et al. (1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138: 863–870. 19. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, et al. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139: 4252–4263. 20. Gould JC, Leonard LS, Maness SC, Wagner BL, Conner K, et al. (1998) Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiol. Mol Cell Endocrinol 142: 203–214. 21. Richter CA, Taylor JA, Ruhlen RL, Welshons WV, Vom Saal FS (2007) Estradiol and Bisphenol A stimulate androgen receptor and estrogen receptor gene expression in fetal mouse prostate mesenchyme cells. Environ Health Perspect 115: 902–908. 22. Okuda K, Takiguchi M, Yoshihara S (2010) In vivo estrogenic potential of 4methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, an active metabolite of bisphenol A, in uterus of ovariectomized rat. Toxicol Lett 197: 7–11. 23. Yoshihara S, Mizutare T, Makishima M, Suzuki N, Fujimoto N, et al. (2004) Potent estrogenic metabolites of bisphenol A and bisphenol B formed by rat liver S9 fraction: their structures and estrogenic potency. Toxicol Sci 78: 50–59. 24. Eiler S, Gangloff M, Duclaud S, Moras D, Ruff M (2001) Overexpression, purification, and crystal structure of native ER alpha LBD. Protein Expr Purif 22: 165–173. 25. Huey R, Morris GM, Olson AJ, Goodsell DS (2007) A semiempirical free energy force field with charge-based desolvation. J Comput Chem 28: 1145–1152. 26. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, et al. (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 27. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31: 455–461. 28. Mocklinghoff S, Rose R, Carraz M, Visser A, Ottmann C, et al. (2010) Synthesis and crystal structure of a phosphorylated estrogen receptor ligand binding domain. Chembiochem 11: 2251–2254. 29. Cheng T, Li X, Li Y, Liu Z, Wang R (2009) Comparative assessment of scoring functions on a diverse test set. J Chem Inf Model 49: 1079–1093. 30. Wang R, Lai L, Wang S (2002) Further development and validation of empirical scoring functions for structure-based binding affinity prediction. J Comput Aided Mol Des 16: 11–26. 31. Neudert G, Klebe G (2011) DSX: a knowledge-based scoring function for the assessment of protein-ligand complexes. J Chem Inf Model 51: 2731–2745. 32. Velec HF, Gohlke H, Klebe G (2005) DrugScore(CSD)-knowledge-based scoring function derived from small molecule crystal data with superior recognition rate of near-native ligand poses and better affinity prediction. J Med Chem 48: 6296–6303. PLOS ONE | www.plosone.org 33. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, et al. (1997) Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389: 753–758. 34. Warnmark A, Treuter E, Gustafsson JA, Hubbard RE, Brzozowski AM, et al. (2002) Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor alpha. J Biol Chem 277: 21862–21868. 35. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, et al. (1998) The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95: 927–937. 36. Tanenbaum DM, Wang Y, Williams SP, Sigler PB (1998) Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains. Proc Natl Acad Sci U S A 95: 5998–6003. 37. Baker ME, Chang DJ (2009) 3D model of amphioxus steroid receptor complexed with estradiol. Biochem Biophys Res Commun 386: 516–520. 38. Baker ME, Chang DJ, Chandsawangbhuwana C (2009) 3D model of lamprey estrogen receptor with estradiol and 15alpha-hydroxy-estradiol. PLoS One 4: e6038. 39. Katzenellenbogen JA (2011) The 2010 Philip S. Portoghese Medicinal Chemistry Lectureship: addressing the ‘‘core issue’’ in the design of estrogen receptor ligands. J Med Chem 54: 5271–5282. 40. Seo JW, Comninos JS, Chi DY, Kim DW, Carlson KE, et al. (2006) Fluorinesubstituted cyclofenil derivatives as estrogen receptor ligands: synthesis and structure-affinity relationship study of potential positron emission tomography agents for imaging estrogen receptors in breast cancer. J Med Chem 49: 2496– 2511. 41. Celik L, Lund JD, Schiott B (2007) Conformational dynamics of the estrogen receptor alpha: molecular dynamics simulations of the influence of binding site structure on protein dynamics. Biochemistry 46: 1743–1758. 42. Manas ES, Unwalla RJ, Xu ZB, Malamas MS, Miller CP, et al. (2004) Structure-based design of estrogen receptor-beta selective ligands. J Am Chem Soc 126: 15106–15119. 43. Nettles KW, Bruning JB, Gil G, O’Neill EE, Nowak J, et al. (2007) Structural plasticity in the oestrogen receptor ligand-binding domain. EMBO Rep 8: 563– 568. 44. DuSell CD, Umetani M, Shaul PW, Mangelsdorf DJ, McDonnell DP (2008) 27hydroxycholesterol is an endogenous selective estrogen receptor modulator. Mol Endocrinol 22: 65–77. 45. Minutolo F, Macchia M, Katzenellenbogen BS, Katzenellenbogen JA (2011) Estrogen receptor beta ligands: recent advances and biomedical applications. Med Res Rev 31: 364–442. 46. Lappano R, Recchia AG, De Francesco EM, Angelone T, Cerra MC, et al. (2011) The cholesterol metabolite 25-hydroxycholesterol activates estrogen receptor alpha-mediated signaling in cancer cells and in cardiomyocytes. PLoS One 6: e16631. 47. Heery DM, Kalkhoven E, Hoare S, Parker MG (1997) A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387: 733–736. 48. McKenna NJ, O’Malley BW (2002) Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108: 465–474. 49. Smith CL, O’Malley BW (2004) Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25: 45–71. 50. Jeyakumar M, Carlson KE, Gunther JR, Katzenellenbogen JA (2011) Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities. J Biol Chem 286: 12971–12982. 51. Billon-Gales A, Krust A, Fontaine C, Abot A, Flouriot G, et al. (2011) Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing. Proc Natl Acad Sci U S A 108: 13311–13316. 52. McInerney EM, Rose DW, Flynn SE, Westin S, Mullen TM, et al. (1998) Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev 12: 3357–3368. 53. Voegel JJ, Heine MJ, Tini M, Vivat V, Chambon P, et al. (1998) The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways. EMBO J 17: 507–519. 54. Hall JM, McDonnell DP, Korach KS (2002) Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements. Mol Endocrinol 16: 469–486. 55. Hall JM, Korach KS (2002) Analysis of the molecular mechanisms of human estrogen receptors alpha and beta reveals differential specificity in target promoter regulation by xenoestrogens. J Biol Chem 277: 44455–44461. 15 October 2012 | Volume 7 | Issue 10 | e46078