Exploring Interactions of Endocrine-Disrupting Compounds with

Chem. Res. Toxicol. 2008, 21, 2195–2206

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Exploring Interactions of Endocrine-Disrupting Compounds with Different Conformations of the Human Estrogen Receptor r Ligand Binding Domain: A Molecular Docking Study Leyla Celik, Julie Davey Dalsgaard Lund, and Birgit Schiøtt* iNANO and inSPIN Centers, Department of Chemistry, UniVersity of Aarhus, DK-8000 Aarhus, Denmark ReceiVed July 30, 2008

Endocrine-disrupting compounds (EDCs) accumulating in nature are known to interact with nuclear receptors. Especially important is the human estrogen receptor R (hERR), and several EDCs are either known or suspected to influence the activity of the ligand-binding domain (LBD). We here present a comparative docking study of both well-known hERR ligands and small organic compounds, including selected polychlorinated biphenyls (PCBs), plasticizers, and pesticides, that are all potentially endocrinedisrupting, into different conformations of the hERR LBD. Three newly found quasi-stable structures of the hERR LBD are examined along with three crystallographic conformations of the protein, either the apo structure or using a protein structure with a bound agonist or antagonist ligand. The possible interactions between the protein and the potentially EDCs are described. It is found that most suspected EDCs can bind in the steroid binding cavity, interacting with at least one of the two hydrophilic ends of the steroid binding site. DDE, DDT, and HPTE are predicted to bind most strongly to the hERR LBD. It is predicted that these compounds can interact with the three conformations of hERR LBD with comparable affinities. The metabolic hydroxylation of aromatic compounds is found to lead to an increase in the binding affinity of PCBs as well as DDT. Docking into the quasi-stable conformations of the hERR LBD leads to computed binding affinities similar to or better than those calculated for the three X-ray structures, revealing that the new structures may be of importance for assessing the function of the influence of EDCs on nuclear receptors. Introduction The human estrogen receptor R (hERR) belongs to the family of nuclear receptors. It is involved in cell specification and activation of transcription, and it is responsible for the female reproductive system (1-6). Two domains are particularly important for nuclear receptor functioning; an N-terminal DNAbinding domain and a C-terminal ligand-binding domain (LBD). The DNA-binding domain has a constitutive activity, whereas the LBD must be activated by binding of a steroid ligand for correct function. Along with a ligand, the hERR LBD also binds coregulator proteins to exert its function (7). Only a part of the coactivator protein is necessary for activation of the hERR LBD. As little as an 8-mer peptide containing a conserved LXXLL motif has been shown to induce activity of the hERR LBD (8). The ligand-dependent activity of the hERR is strongly correlated to the conformation of the LBD (9). X-ray crystallography of the nuclear receptors has shown three general conformations of the LBD of nuclear receptors: the apo, agonist, and antagonist conformations, where the names refer to the type of bound ligand. Each of the conformations shows a different position of helix 12 (H12), often referred to as the activation factor 2 helix (2, 5, 6). The change in position of H12 is believed to direct the recognition of coregulator proteins by the specific 1

* To whom correspondence should be addressed. Tel: +45 8942 3953. Fax: +45 8619 6199. E-mail: [email protected]. 1 Abbreviations: hERR, human estrogen receptor R; DHT, dihydrotestosterone; E2, estradiol; E3, estriol; EDC, endocrine-disrupting compound; FUL, fulvestrant; H12, helix 12; LBD, ligand-binding domain; MD, molecular dynamics; OHT, 4-hydroxytamoxifen; PCB, polychlorinated biphenyl; RAL, raloxifen; rmsd, root-mean-square deviation; SERM, selective estrogen receptor modulator; ZEA, zearalenone.

conformation of hERR LBD, hereby controlling the function of the receptor as being either active, with an agonist bound, or inactive, with an antagonist bound (10). The steroid-binding site of the hERR LBD is highly hydrophobic and thereby adapted to binding of the endogenous steroid ligand estradiol (E2, Figure 1). However, the two ends of the binding site are made up of three ionizable residues, Glu353, Arg394, and His524; the first two residues are positioned in one end of the binding site, the latter in the other. These three residues are the only charged or polar residues in the binding site, and together, they function to anchor E2 through several hydrogen bonds. Because the position of hydrogen atoms cannot easily be deduced from protein crystallography, the charge and tautomer state of His524 in the binding site are not known per se. In a recently published molecular dynamics (MD) study, we have shown that His524 is most likely to be uncharged and found as the -tautomer (hydrogen atom placed on N) when E2 is bound in the active agonist conformation of the hERR LBD. A hydrogen bond is thus found where the E2 O17 hydroxyl group functions as the hydrogen bond donor and His524 Nδ as the hydrogen bond acceptor (11). In an X-ray structure of the hERR LBD in the antagonist conformation with bound 4-hydroxytamoxifen (OHT, Figure 1), the side chain of His524 is rotated as compared to the agonist conformation, yielding a structure where His524 is not interacting with the OHT unsubstituted phenyl group. OHT is a selective estrogen receptor modulator (SERM), and the phenyl group is located in the same position as the steroid D ring in the crystal structure (12). When an antagonist ligand bearing a potential hydrogen bond donor is bound, His524 is still rotated away from the original position; however, raloxifene (RAL) is able to form a

10.1021/tx800278d CCC: $40.75  2008 American Chemical Society Published on Web 10/16/2008

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Figure 1. Structure of ligands used for the docking studies; hERR agonist and antagonist ligands, nonestrogenic steroids, and phytoestrogens.

hydrogen bond to His524 (13). Along with data from the MD simulations (11), it was suggested that a proton shift of His524 may occur during activation of the hERR LBD when changing from the apo conformation to the agonist conformation upon steroid binding. By binding to the hERR LBD, endocrine-disrupting compounds (EDCs) can disturb the physiological functions of the receptor and cause various serious diseases (14-16). Studies have shown that when complexed with E2 or OHT, the hERR LBD may interact with different coregulator peptides, while binding of, for example, some pesticides leads to interactions with yet other peptides not necessarily containing the conserved LXXLL motif (17-19). On the basis of these observations, novel conformations of the hERR LBD have been suggested to be present in these complexes. Because of their possible interactions with nuclear receptors, several classes of compounds have been modeled in the binding site of different LBDs. Polychlorinated biphenyls (PCBs), DDT, and some of its metabolites have previously been docked in the hERR LBD (20), revealing that a single binding mode for EDCs was not easy to obtain, even though only the agonist conformation of the hERR was included. Free energy perturbation calculations have also been performed on PCBs; this study only includes a single binding mode, aligning the aromatic skeleton with E2 in the binding site, and depicts how the ligand samples different rotamer states during MD simulations (21). While comparative docking studies of the androgen receptor have discussed endocrine-disrupting properties of a diverse set of compounds (22, 23), the hERR LBD has mostly served as the system of choice for testing new computational approaches (24-30) or in the development of novel SERMs (31-36). In our MD simulation study, we have observed new stable conformations of the hERR LBD on the nanosecond time scale (11), which may be of relevance for the binding of pesticides and other compounds resulting in the new recognition pattern of coregulator peptides as suggested from experimental studies (17-19). On the basis of results from our MD simulations (11), the different charge and tautomer states as well as the orientation of His524 may also be of importance for binding of other ligands such as EDCs. Two hERR agonist ligands (E2 and estriol, E3), two SERMs, OHT and RAL that are both partial antagonists of the hERR LBD, as well as a full antagonist ligand, fulvestrant (FUL), all depicted in Figure 1, were docked into the three known X-ray structures of the hERR LBD and four selected MD snapshot structures. The snapshots each represent a conformation of the

hERR LBD that is different from the crystal structures. They were thereby differentiated, and the properties of the binding sites were compared to those of the three well-known X-ray structures (12, 13, 37). Three of the snapshot structures represent new quasi-stable conformations identified in our recent MD simulation study of the hERR LBD; one originates from an antagonist structure, called 6P, indicating a protonated His524, and two stem from an apo simulation, called 7E (N tautomer of His524) and 7D (Nδ tautomer of His524), respectively; the fourth snapshot represents the active agonist conformation of the hERR LBD, called 1E (Figure 2) (11). The latter structure is included in this study to serve as an internal reference to evaluate the effect of performing an MD simulation on a protein structure prior to docking. Three nonestrogenic steroids and three phytoestrogens were also docked (Figure 1). Five biphenyl compounds and eight plasticizers (Figure 3), all suspected of endocrine-disrupting functions, were included, as well as 20 pesticides (Figure 4). The majority of the pesticides have previously been found relevant regarding the activity of the hERR LBD, either by peptide-binding studies (19) or in studies measuring binding affinities to the hERR LBD (26, 27, 38-40). Some of the pesticides are no longer used in large scales (e.g., DDT, which has been banned for decades), and some plasticizers must be avoided in specific applications (e.g., bisphenol A in baby bottles). Despite this, they are all relevant to include to understand the function of EDCs with regard to the hERR. It is generally problematic to determine endocrine-disrupting potencies, and research activities addressing these questions are abundant (41-43), affecting political legislation worldwide (44-46). The compounds depicted in Figures 1, 3, and 4 were docked into the seven structures of the hERR LBD to obtain knowledge of preferences for binding into the steroid-binding site in specific conformations of the hERR LBD, taking the three possible charge and tautomer states of His524 into account.

Experimental Procedures Three X-ray structures of the hERR LBD and four quasi-stable structures identified from MD simulation were included in this study. The three X-ray structures comprised an artificial apo conformation of the hERR LBD with bound E2 [pdb entry 1A52 (37)], an agonist conformation of the hERR LBD with bound E2 [pdb entry 1GWR (47)], and an antagonist conformation of the hERR LBD with bound OHT [pdb entry 3ERT (12)]. The three possible charge and tautomer states (abbreviated as Nδ, N, and Nδ/N for the charged histidinium ion) of His524 were considered for the three X-ray structures resulting in nine setups.

Docking of EDCs in Various hERR LBD Conformations

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Figure 2. Structure of the three hERR LBD X-ray structures (panels A, C, and G) and four snapshots from MD simulations (panels B and D-F) included in this docking study. The core of the LBD is shown in gray; H11 and H12, the two helices where the structures differ, are colored purple; and the coactivator peptide included in the agonist conformation is yellow.

Figure 3. Structure of PCBs, phthalates, and other plasticizers included in the docking study.

The four snapshots were identified from our recent study on MD simulations of the hERR LBD; all had E2 present in the binding site (11). Snapshot 1E was extracted from a simulation of the agonist conformation of the hERR LBD with a bound coactivator peptide (pdb entry 1GWR) after 1.5 ns with His524(N). Similarly, 6P was taken after 5.0 ns from a simulation of the hERR LBD antagonist conformation (pdb entry 3ERT) with His524(Nδ/N), 7D was taken after 5.0 ns simulation of the hERR LBD apo conformation (pdb entry 1A52) with His524(Nδ), and 7E was taken after 8.0 ns simulation of the hERR LBD apo conformation (pdb entry 1A52) with His524(N) (11). The nomenclature follows the one used in the original description of the simulations. 1E was included here to examine the effects of an MD simulation on the structure of the binding site and to see the effect of including some protein flexibility before the docking calculation was carried out, when comparing the use of the static X-ray structures. The three latter snapshots, on the other hand, all showed changes in the position of H12 and had modified structures of the binding site.

For the four snapshot structures, the protonation and tautomer state of His524 were kept the same as in the original MD simulations, entailing a total of 13 protein structures for docking. Ligand Preparation. All ligands (Figures 1, 3, and 4) were drawn in Maestro 7.5 (48) and minimized in vacuum with a Conjugate Gradient algorithm in MacroModel 9.1 (48) employing the MMFFs (49) force field. Protein Preparation. For the agonist and apo (pdb entries 1GWR and 1A52) structures of the hERR LBD, only chain A was included in this docking study. The antagonist conformation of the hERR LBD was modeled from pdb entry 3ERT. Protonation states and binding orders were checked for all ionizable amino acids, and a structure was created for each of the three His524 charge and tautomer states. The ProteinPreparation application of Impact 4.0 (48) was employed with the “preparation and refinement” option to add missing atoms and remove induced steric strain after the addition of hydrogen atoms in all structures. The “preparation” part of this application assigned protonation states of ionizable residues

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Figure 4. Structures of pesticides included in this study, divided into subclasses.

based on the local environment, optimized the orientation of polar side chains, and attempted to neutralize the system. During the “refinement” part, the protein was energy minimized to a maximum root-mean-square deviation (rmsd) of 0.3 Å. Conformations from MD simulations already contained all atoms and were prepared using the “refinement only” option to optimize the structure of the protein. Residues Glu353 and Arg394, both located close to the steroid A ring in the X-ray structures, were modeled as charged in all setups, as was Asp351, located near the alkylamine functional group of OHT in the channel leading toward the surface of the hERR LBD in the antagonist conformation of the protein (12). A water molecule positioned near Glu353 and Arg394 in the agonist and antagonist structures was included in setups of the two conformations, as was a corresponding water molecule found in the MD snapshots. In the 6P snapshot, three water molecules, located in the other end of the binding site (near His524) during the MD simulation, were similarly included. One of these substituted for His524, which was rotated out of the binding site in this simulation; the two other water molecules helped form a water-wire to the surface of the protein. No water molecule was present between Glu353 and Arg394 in the apo conformation of the hERR LBD and was therefore not included in these setups. Docking. Grids for all protein setups were calculated in Glide 4.0 (48, 50) with the binding site defined from the bound ligand, that is, E2 for the apo and agonist conformations and OHT for the antagonist conformation. All ligands were docked flexibly into the 13 protein structures using Glide 4.0 with default settings (48, 50), saving one pose for each ligand and using the standard precision scoring function. MD Simulation. Stochastic MD simulations were performed of HPTE in the agonist conformation and of DDT in the agonist and antagonist conformations of the hERR LBD. The simulations were performed in MacroModel 9.1 (48) with the implemented OPLS2005 force field (51, 52), all with His524 modeled as the -tautomer. The OPLS force field was parametrized for protein-ligand systems

(51) and it is comparable to other protein force fields (53, 54). The complexes were simulated for 1 ns with 1 fs time step at 300 K with an implicit GB/SA water model as implemented in MacroModel 9.1 (48, 55). Finally, snapshots of the trajectories were written every picosecond.

Results Docking of Estrogens and SERMs in X-ray Structures. The hERR ligands along with selected steroids and phytoestrogens (Figure 1) were initially docked in the three X-ray structures examining the effect of the possible charge and tautomer state of His524. We want to emphasize the importance of taking all possibilities for the charge and tautomer state into account for histidine residues in modeling studies, to fully account for the missing information from X-ray diffraction, and to capture all possibilities of ligand binding. To obtain an internal measure of the accuracy of the applied methodology using Glide, it is important to first check how well cocrystallized ligands can be redocked into the prepared native protein structures. It is possible to redock E2 in the apo and agonist structures with high accuracy, as measured by a low rmsd of 0.2-0.3 Å (Table 1). The redocking of OHT to the prepared protein of 3ERT results in a slightly decreased accuracy, and the rmsd is found to be higher at 1.2-1.6 Å, with the primary differences being in the position of the substituted amine. These computed numbers for redocking of cocrystallized ligands validate the chosen method and show that the physicochemical properties of the steroid binding cavity of the hERR LBD are accounted for satisfactorily using this methodology. The two estrogens, E2 and E3, both show a preference for docking into the agonist and apo conformations, with the two being comparable in measured G-scores. For the antagonist



Table 1. Computed G-Score in kcal/mol for Docking of hERr Agonist and Antagonist Ligands into Three X-ray Structures and Four MD Snapshots of the hERr LBD apo Nδ apo N apo Nδ/ agonist Nδ agonist N agonist Nδ/ antagonist Nδ antagonist N antagonist Nδ/ 1E (agonist) 6P (antagonist) 7D (apo) 7E (apo)

E2

E3

OHT

-10.96 -10.83 -10.73 -11.39 -11.13 -11.24

-10.64 -11.50 -10.45 -11.25 -11.62 -11.10 -5.82b -6.02b -5.81b -10.59 -10.64 -10.25 -10.46

-10.25 -10.30 -9.66

-5.50b -10.15 -10.07 -10.48 -10.57

RAL

FUL -5.42

-10.76 -9.51 -7.42 -10.98 -9.40 -6.51 -10.90 -8.96 -5.28 -6.36 -8.42

rmsd (Å)a 0.32 0.23 0.23 0.22 0.22 0.22 1.55 1.26 1.27 0.80 1.39 1.88 1.96

a rmsd values are calculated for E2 in apo and agonist setups as well as in the MD snapshots and for OHT in the antagonist setups. b These poses either do not place the ligand in the binding site or rotate the ligand as compared to the standard orientation. Blank fields imply that no poses were generated.

binding site, on the other hand, the estrogen compounds bind very poorly and in fact not in the steroid binding site. Instead, in the collected poses, the ligand is placed in a channel leading from the steroid binding site to the surface of the protein, which in 3ERT, the antagonist structure, is occupied by the alkylamine side chain of OHT. Binding of E2 and E3 involves hydrogen bonds from the steroid O3 (see Figure 1 for numbering of important atoms in E2) to Glu353, Arg394, and the structural water molecule (see Figure 5). The steroid O17 is hydrogen bonded to His524(Nδ). The binding of E2 is relatively unaffected by the protonation state of His524. E3, however, shows a marked preference for the -tautomer; in this state, both of the D ring hydroxyl groups are able to form hydrogen bonds to His524(Nδ). This is in fine agreement with our recently published MD study where the -tautomer of His524 was found to be the preferred state for formation of the active conformation of the hERR LBD (11). OHT docks relatively similar in both the apo and the antagonist conformations of the hERR LBD. RAL can only dock in the latter, and FUL is poorly scored in both the apo and the antagonist conformations (Table 1). Both OHT and RAL form hydrogen bonds to Glu353 and Arg394 in the binding site and to Asp351 in the binding pocket, accommodating the alkylamine side chain of OHT in the antagonist structure. RAL furthermore forms a hydrogen bond with the backbone carbonyl group of Gly420, located near His524, and shows a preference for binding to the hERR LBD with a neutral tautomer of His524, with the Nδ and N tautomers scoring similar (see Figure 5). Binding of FUL in the antagonist conformations of the hERR LBD is similar to binding of E2, although the steroid ring is flipped 180° around the long axis of the ring system, hereby positioning O17 similar to O16 in E3, allowing the long aliphatic chain to protrude through the same channel as the OHT and RAL alkylamine side chains. The rotation of the steroid skeleton is possibly caused by the position of the aliphatic chain in FUL, which is attached to C7 of the steroid skeleton. If the steroid skeleton was to bind to the hERR LBD in the same orientation as is seen for E2, one can speculate that it would be more favorable to attach the aliphatic chain on the C ring or maybe as the other enantiomer. Hydrogen bonds are present between the FUL O3 hydroxyl group and Glu353 and Arg394 as well as between the O17 hydroxyl group and Gly420 and possibly to His524 when this residue is modeled as the -tautomer. The end of the aliphatic arm is fully exposed at the surface of the protein in all generated poses.

Docking of Estrogens and SERMs in Structures from MD Simulations. In the employed MD snapshots, Glu353 and Arg394 have been slightly displaced, as compared to the X-ray structures, forming a bifurcated salt bridge. As a result, Arg394 no longer participates in hydrogen bonds to the ligands. This is reflected in slightly poorer computed G-scores due to the missing electrostatic interaction (Table 1). After docking to the 1E (agonist), 7D, and 7E (both apo) structures, E2 and E3 are bound similarly to what is observed in the X-ray structures with hydrogen bonds to Glu353, His524, and the structural water molecule located next to Glu353 and Arg394. In 6P (antagonist), on the other hand, His524 is rotated, which is a general observation for the structures with bound antagonists. As a consequence, His524 can no longer participate in a hydrogen bond to the estrogen ligands. Instead, a water molecule, which was found in the MD simulation, is included here, interacting with the E2 O17 hydroxyl group, Glu419, and the two other water molecules. It is interesting to notice that the computed G-scores for the ligands are slightly poorer when docked in the snapshot structures than when using the X-ray structures directly in the docking calculations (Table 1). This may be due to the lack of a hydrogen bond to Arg394. RAL and FUL can be docked in neither of the MD structures, but OHT is able to dock in 7D and 7E, although rather poorly (Table 1). This demonstrates that the differences between the original X-ray structure, where OHT binds quite well, and the MD structures are of importance for ligand binding. It may further be speculated that the differences in size between OHT and both RAL and FUL can explain why only the former can dock into the apo conformation. Docking of Nonestrogenic Steroids and Phytoestrogens. Docking of three nonestrogenic steroids (see Figure 1) demonstrates the importance of having a hydrogen bond donor on the A ring and a hydrogen bond donor or acceptor in the proximity of the D ring. Progesterone and R5020, an endogenous and a synthetic progestogen, both have hydrogen bond acceptors at these two positions. It is only possible to obtain poses for these two progestogens in two of the four snapshots (6P and 7D), and there is no consistency in the orientation of the steroid skeleton. The computed G-scores (Table 2) are rather poor as compared to E2 and E3. It may be speculated that the larger groups attached at the D ring in the progestogens are the reason for the fact that no poses are found in most setups: They are too large to fit in the steroid binding site. For dihydrotestosterone (DHT, see Figure 1), poses are generated for all but the antagonist setups, revealing that the smaller group on the D ring is preferred. Furthermore, the computed G-scores are considerably better (Table 2) than those measured for the progestogens, although they are still significantly poorer than for E2, being around -7 kcal/mol as compared to approximately -11 kcal/mol for the natural ligand. The DHT A ring carbonyl group is hydrogen bonded to Arg394 and the structural water molecule, while the D ring hydroxyl group interacts with His524 in all poses. This binding appears to be most favorable in the 1E and 7D snapshots and least favorable in the apo conformation. Similarly to the results for E2 and E3, no poses of DHT are found in the antagonist conformation. Because DHT structurally binds similarly in the 10 protein setups, where poses are generated, the observed differences in the estimated binding affinity appear to originate from small differences in the binding site structure. It is here worth noticing that DHT is an endogenous androgen hormone, but it does have the ability to bind to the hERR with a relative binding affinity of 0.03% as compared to E2 (26), which relates to a difference in binding

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Figure 5. Ligand binding in the hERR LBD. (A) E2 in the agonist, (B) OHT in the apo, and (C) RAL in the antagonist conformations. All structures are modeled with His524 as the -tautomer with the N hydrogen atom pointing into the figure. Residues important for interactions between the hERR LBD and the ligands are displayed in all panels, and the structural water molecule is included in the agonist and antagonist conformations.

Table 2. G-Scores (in kcal/mol) for Docking of Nonestrogenic Steroids and Phytoestrogens into Three X-ray Structures and Four MD Snapshots of the hERr LBD progesterone R5020 DHT genistein coumestrol ZEA Apo Nδ Apo N Apo Nδ/ agonist Nδ agonist N agonist Nδ/ antagonist Nδ antagonist N antagonist Nδ/ 1E (agonist) 6P (antagonist) 7D (apo) 7E (apo)

-7.20 -7.57 -7.18 -8.22 -7.76 -8.19

-7.72 -9.12

-9.19 -7.80 -8.63 -7.13 -9.51 -7.41

-9.18 -8.62 -8.81 -8.74 -8.95 -8.83 -8.93 -8.85 -8.91 -9.24 -8.01 -9.23 -8.26

-9.10 -9.10 -8.92 -9.32 -9.91 -9.51 -8.66 -8.02 -8.78 -8.95 -9.12 -9.67 -9.46

-5.91 -5.92 -5.90

affinities of 4-5 kcal/mol in nice accordance with the computed difference in G-scores. Genistein and coumestrol, two phytoestrogens, are structurally similar to the estrogens, with two hydroxyl groups at opposite ends of a (partly hydrophilic) isoflavone skeleton. Both phytoestrogens are able to dock in all 13 protein setups with G scores of approximately -9 kcal/mol (Table 2). In general, the phytoestrogens are found in two different orientations in the collected poses, interchanging the two ends of the molecules relative to the two polar ends of the binding site. For genistein, a rotation around the long axis of the molecule seems insignificant, since this only interchanges the position of two hydroxyl groups on the “A ring”. The rotation also swaps the positions of the ether and carbonyl group of B-ring; these two are in a hydrophobic part of the hERR LBD binding pocket, and the rotation does not affect any possible interactions. Zearalenone (ZEA), a mycotoxin found in moldy corn (56), is only able to dock in the antagonist conformation of the hERR LBD and with relative poor G-scores (-5.9 kcal/mol, see Table 2). It is placed in the channel leading toward the surface of the LBD and possibly interacts with the Asp351 carboxylate group, the Thr347 carbonyl group, and the Cys530 and Leu536 backbone amide groups. Only the interaction to Asp351 may be characterized as a hydrogen bond; the other interactions show long oxygen to hydrogen distances (2.6-4.3 Å). Docking of Polychlorinated Biphenyls. The PCBs are able to dock in all protein setups, except for PCB169, where no poses are generated for docking in setups 1E and 6P (Table 3). They generally bind similarly to E2 with the aromatic para-substituents pointing toward the two hydrophilic ends of the binding site (see Figure 6), in accordance with a study by Ostenbrink et al. (21). For monohydroxylated PCBs, the hydroxyl group is interacting with Glu353 and Arg394; in PCB1-4,4′-diOH, the

second hydroxyl group mimics the E2 O17 hydroxyl group and interacts with His524. The hydroxylated PCBs generally bind better to hERR LBD than the nonhydroxylated ones, with G-scores in the range of -9 to -7 kcal/mol as compared to -7 to -4 kcal/mol for the latter. There is no significant difference between reported G-scores of the monohydroxylated (PCB10-4′-OH) and dihydroxylated PCBs (PCB1-4,4′-diOH), showing that the second hydroxyl group is not required for improving the binding affinity as compared to possible hydrophobic interactions. The hydroxylated PCBs show a small preference for binding in the X-ray conformations (G-score < -8 kcal/mol) to the MD snapshots (G-score > -8 kcal/mol). PCB153 and PCB169, both lacking hydroxyl groups to anchor them in the binding site, are placed in the channel leading toward the protein surface in the antagonist hERR LBD conformations. This may be due to the tighter space found in the channel as compared to the steroid binding site, allowing for a larger number of hydrophobic interactions. Docking of Plasticizers. Phthalates show a very unspecific style of binding in the binding cavity of the hERR LBD in these docking simulations. In the antagonist setups, there is a tendency for binding in the channel leading toward the surface of the protein. In the remaining 10 setups, the compounds are scattered around in the binding site, mainly with hydrophobic interactions to the protein (see Figure 7). Despite the two ester groups present in all phthalates, only a few are found to form hydrogen bonds to the protein; the hydrogen bond donor is then primarily His524. Another possible hydrogen bond can be formed to the water molecule located near His524 in the 6P structure. The G-score for phthalates range between -8 and -1 kcal/mol (Table 3), and the E-model (not shown), which includes contributions of the internal energy of the ligand in the score, is calculated to be between -65 and 5 kcal/mol, revealing that a great deal of steric strain may be present in some of the poses. The phthalates with short side chains (BBP and DBP) generally dock better than those with longer aliphatic side chains. The phthalates show reversed electrostatic properties as compared to the steroids and phytoestrogens, being polar in the middle with hydrophobic ends, which may rationalize their poor docking scores. Bisphenol A, a plasticizer known for its estrogenic activity (16), docks in all 13 protein setups (Table 3) with a hydrogen bond between one of the phenol groups and Glu353. It is also possible to form hydrogen bonds with Arg394 and the structural water molecule, although these are only present in a few poses. Three poses (from snapshots 1E, 7D, and 7E) show a hydrogen bond from the second phenol group to His524, but mostly, this phenol is interacting with the Thr347 hydroxyl group (in the



Table 3. G-Scores (in kcal/mol) for Docking of Potential EDCs into Three X-ray Structures and Four MD Snapshots of the hERr LBDa apo conformation Nδ

N

Nδ/

agonist conformation Nδ

N

antagonist conformation

Nδ/

N

Nδ/

1E

6P

7D

7E

-8.74 -8.36 -8.42 -5.25 -6.00

-8.76 -8.39 -8.32 -5.27 -6.02

-8.75 -8.37 -8.45 -5.26 -5.99

-7.88 -7.73 -7.89 -7.12

-8.00 -7.68 -7.93 -5.44

-7.74 -7.69 -7.66 -7.42 -7.05

-8.04 -7.90 -7.78 -7.39 -6.06

plasticizers -7.20 -7.28 -4.00 -3.56 -3.93 -4.01 -3.78

-6.77 -3.39 -4.98 -3.95 -5.72

-6.15 -3.57 -4.60 -3.53 -3.82

-8.03 -3.98

-5.11 -4.22

-7.65 -3.97

-7.22 -3.79

biphenyls -8.42 -8.91 -8.34 -7.36 -4.86

PCB10-4′-OH PCB1-4,4′-diOH PCB4-4′-OH PCB153 PCB169

-8.20 -8.28 -8.17 -6.87 -5.33

-8.17 -8.23 -8.03 -6.73 -4.86

-8.14 -8.28 -7.99 -6.96 -5.12

-8.48 -8.96 -8.46 -7.42 -5.00

-8.58 -8.56 -8.34 -7.32 -5.13

BBP DBP DEHP (R,R) DEHP (S,S) DEHP (R,S) DiNP DiDP bisphenol A nonylphenol

-6.77 -3.56 -3.79

-6.35 -3.69

-6.72 -3.67

-7.31 -3.81

-3.82 -1.32 -8.67 -2.27

-4.15

-6.40 -3.32 -3.60 -4.16 -2.96 -4.21

-7.79 -3.34

-8.65 -1.92

-7.36 -3.23

-7.71 -3.13

-7.37 -3.08

atrazine chlormequat chlorothalonil chlorpyrifos DDE DDT deltamethrin daminozide dichlorvos dieldrin dimethoate endosulfan fenarimol fenpropathrin HPTE iprodione methomyl pirimicarb propamocarb tolchlofosmethyl

-6.23 -4.64 -6.95 -6.59 -7.00 -7.44

-6.05 -4.36 -6.17 -6.29 -7.13 -7.34 -4.83 -3.94 -4.55 -6.72 -5.99 -6.48 -6.66 -2.39 -9.00 -6.64 -3.78 -6.59 -0.78 -6.48

-6.22 -4.75 -6.85 -6.33 -6.96 -7.33 -4.65 -4.34 -5.26 -6.78 -5.84 -6.38 -6.45 -4.43 -9.05 -5.77 -4.00 -6.75 -0.58 -7.21

-5.30 -4.75 -6.88 -6.99 -6.99 -5.36

-5.57 -4.58 -6.12 -6.12 -6.75 -5.56

pesticides -5.58 -4.39 -6.81 -6.51 -7.00 -5.46

-5.01 -5.32 -6.89 -6.93 -6.83 -6.36 -5.97 -8.94 -5.94 -4.28 -6.82 -0.64 -7.27

-4.61 -4.80 -6.74 -5.82 -6.91 -6.40

a

-4.40 -5.36 -6.86 -5.93 -6.64 -6.45 -2.57 -8.91 -6.52 -3.77 -6.73 -0.66 -7.35

snapshots from MD simulations

Nδ

-3.08

-9.12 -4.85 -4.29 -6.51 -1.02 -6.47

-4.57 -5.20 -6.81 -6.82 -6.75 -5.57 -5.55 -8.04 -5.69 -4.38 -6.87 -0.61 -7.21

-8.06 -2.73

-8.11 -2.83

-8.12 -2.76

-7.72 -3.01

-7.18 -3.16

-7.94 -3.35

-4.14 -7.61 -2.77

-5.61 -3.70 -5.03 -4.33 -7.32 -8.05 -5.87 -4.04 -4.78 -5.74 -6.05 -5.20 -5.89 -4.88 -8.86 -7.46 -4.53 -5.67 -1.21 -6.40

-6.01 -3.72 -4.89 -5.65 -7.31 -8.05 -5.92 -4.77 -4.85 -5.45 -6.03 -5.02 -5.93 -4.86 -9.04 -6.66 -4.59 -5.89 -1.06 -6.47

-5.64 -3.68 -5.03 -5.08 -7.32 -8.06 -5.90 -4.69 -4.89 -5.69 -6.06 -5.09 -5.87 -5.00 -8.87 -7.46 -4.57 -5.65 -1.20 -6.52

-5.16 -4.79 -6.57 -6.10 -7.19 -7.85

-5.95 -4.03 -6.37 -6.50 -7.30 -7.76 -5.29 -4.00 -4.67 -6.19 -5.30 -5.91 -6.68 -7.09 -7.22 -6.13 -4.68 -6.26 -0.65 -6.32

-5.45 -4.76 -6.59 -7.17 -7.19 -7.54

-6.61 -4.60 -6.82 -6.76 -6.90 -6.14

-4.13 -5.37 -6.91 -5.71 -6.27 -5.35

-4.20 -4.62 -6.87 -5.72 -6.24 -7.10 -4.89 -8.05 -6.57 -4.43 -6.59 -0.87 -6.70

-4.52 -5.14 -6.84 -6.38 -6.24 -5.94 -8.20 -4.82 -6.83 -0.93 -6.38

-7.97 -8.01 -4.16 -7.04 -1.22 -7.26

Abbreviations for PCBs and phthalates are given in Figure 3.

Figure 6. Binding of PCB1-4,4′-diOH in the agonist N setup. All hydroxylated PCBs bind in a similar manner with hydrogen bonds to Glu353 and Arg394.

Figure 7. Binding of the 15 best scoring poses of phthalates (all have G-scores below -5 kcal/mol) shown in the framework of the binding site of hERR LBD agonist N. Binding is generally very scattered.

antagonist and apo setups). A third possibility for the second phenol group is to point toward a hydrophobic part of the binding site (seen in some of the poses generated in the agonist and apo N setups), even though this leads to poorer G-scores due to the lack of a hydrogen-bonding partner to the second hydroxyl group. Nonylphenol, a xeno-estrogen used for softening plastics, can also dock in all 13 protein setups, although with very poor G-scores (Table 3). The phenol group is found to hydrogen bond to Glu353, Arg394, and the structural water molecule in one end of the binding site, mimicking the E2 A ring. The position

of the nonylphenol alkyl side chain is randomly scattered around in the binding site in the poses. Docking of Pesticides. Computed G-scores for the examined pesticides are listed in Table 3. The 20 pesticides bind with very different docking scores ranging from -9 to -0.6 kcal/ mol. HPTE is computed to have the overall best score, and propamocarb (G-score approximately -1 kcal/mol) has the worst score in most hERR LBD models. The majority of pesticides are able to dock in all protein setups. However, only 6% of the collected poses have G-scores below -8 kcal/mol (DDT, HPTE, and iprodione), 65% have G-scores better than

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Celik et al.

Figure 8. Binding of (A) DDT in the antagonist structure, (B) DDT in the agonist structure, (C) HPTE in the agonist structure, and (D) HPTE in the 6P snapshot. In A, B, and C, His524 is modeled as the neutral -tautomer and in D as the charged histidinium ion.

-5 kcal/mol, and only three ligands do not show any poses with G-scores better than -5 kcal/mol (chlormequat, methomyl, and propamocarb). Pesticides with favorable G-scores generally correspond to those giving a DDE-like response in the peptide binding studies by Sumbayev et al., while those with poorer G-scores correspond to pesticides that have a peptide recognition pattern similar to the one found in the apo complex (19). This may imply that these pesticides either bind very poorly or not at all to the hERR LBD or that they accomplish their endocrinedisrupting function through another mechanism. There is a strong tendency for favoring binding modes that include hydrogen bonds or salt bridge interactions to either end of the steroid-binding site. Interactions are, thus, found to either Glu353 and/or Arg394 or to His524. None of the pesticides, though, are able to form hydrogen bonds to both ends of the binding site, which would fully anchor them. A few of the pesticides are placed in the channel leading toward the protein surface when docking in the antagonist hERR LBD conformation. These poses generally have poorer G-scores than the corresponding ones in the steroid binding site. All examined pesticides bind similarly in the different protein setups; this is also reflected in the G-scores (see Table 3). On the basis of the position of the ligand and differences in G-scores, only three pesticides (chlorpyrifos, fenpropathrin, and iprodione) show great variation in their predicted binding affinities across the explored conformations of the hERR LBD. The presence of charged or polar groups on the ligand, on the other hand, does not seem to give any indications about the strength of binding affinities. DDT and its metabolites, DDE and HPTE, are found to bind particularly well to the hERR LBD. They tend to bind in one of two different orientations; all poses have one of the phenyl rings pointing toward Glu353 and Arg394, thus mimicking the

steroid A ring, while the other phenyl ring can either point toward the channel leading to the surface and Thr347 (for the antagonist protein conformation) or into the bottom of the hydrophobic part of the steroid binding pocket, hereby interchanging the position of this phenyl ring with the trichloro methyl group (see Figure 8). These binding modes are similar to the ones observed for bisphenol A (see above). Both DDT and HPTE prefer binding with the second phenyl ring pointing toward Thr347 in the channel in all structures. This is in accordance with the observation that a His524Ala mutation essentially does not affect binding of DDE (19). G-scores for HPTE range between -7.24 and -9.12 kcal/mol. The reported differences in G-scores result from minor translations of the molecule in the binding site. The rmsd value for the different poses are between 0.5 Å and 1.5 Å, inducing different geometries for possible hydrogen bonds to Arg394, entailing the observed differences in the computed G-scores. Poses for DDT and its metabolites are generated in all setups, and generally, they all score relatively well. This may indicate that either using this methodology, a discrimination cannot be made or that these molecules can in fact interact with and bind to all of the conformations of the hERR LBD. The compounds would thus have ample opportunities to affect the ensemble of structures, which are present in a delicate conformational equilibrium, as suggested by some researchers (9, 57). Chlorpyrifos is predicted to bind markedly better in the 7D structure (G-score ) -7.17 kcal/mol) and worse in the antagonist Nδ structure (G-score ) -4.33 kcal/mol) as compared to the general binding mode in the sampled structures. This difference can be explained by a possibility to form a hydrogen bond to His524 from one of the phosphoester oxygen atoms, which is found only in the 7D snapshot. Instead, the


dominant binding mode in the remaining protein structures has a polar interaction between the chloride substituents on the aromatic ring and either end of the binding site, while binding in antagonist Nδ reveals neither a hydrogen bond nor the polar interactions. Fenpropathrin has a single pose with a good docking score (6P, G-score ) -7.09 kcal/mol) and two poses with very low docking scores (apo Nδ and apo N setups, G-score ) -2.57 and -2.39 kcal/mol, respectively), while the other poses have G-scores around -5 kcal/mol. In the 6P structure, the ligand is positioned in the middle of the binding site and filling it. A similar binding is observed when fenpropathrin is docked in the 7E snapshot and in the agonist Nδ and Nδ/N setups. In the other setups, fenpropathrin is located either in the channel leading to the surface of the LBD or on the surface of the LBD, leading to the mediocre binding of the ligand. Docking poses of iprodione are found in two different places in the hERR LBD, either in the steroid binding site (for agonist and snapshot structures) or in the channel leading toward the surface (apo and antagonist setups). One of the poses placing iprodione in the steroid binding site has a more favorable G-score than the others (-8.01 kcal/mol); this pose has a cis conformation of the amide group, while all other poses have the energetically more favorable trans conformation of the ligand. The reason for the more favorable binding of the cis conformation is an additional hydrogen bond between iprodione and the Leu387 carbonyl group, which is not possible when the trans conformation of the amide bond is found. MD Simulations. Finally, MD simulations were performed on HPTE in the agonist N protein and on DDT in the agonist N and antagonist N proteins to study whether the predicted binding modes of this ligand are stable. The binding mode for DDT is the same in the antagonist N protein as is found for HPTE, whereas it is rotated in the binding site in the agonist N protein (see above). During the MD simulation, the position of HPTE is maintained with a computed rmsd value for all heteroatoms of ∼2 Å as compared to the minimized docking pose, corresponding roughly to the expected level of thermal fluctuations of the molecule. The hydrogen bond between one of the phenol rings in HPTE and Glu353 is conserved throughout most of the simulation; when this interaction is not present, a hydrogen bond to the carbonyl group in Leu387 is formed instead. The second phenol ring forms a conserved hydrogen bond to the Thr347 hydroxyl group, as was observed in the docked pose. The two setups of DDT in the hERR LBD are also stable during the MD simulations. In the agonist N setup, an rmsd of 1.5-2 Å is observed for the ligand. This mainly corresponds to a rotation of the phenyl ring near His524, revealing that the molecule is not anchored very well in this end of the binding cavity. In the antagonist N setup, the rmsd for DDT is stable at ∼2.1Å, and the ligand binds comparably with HPTE, filling parts of the steroid pocket near Glu353 and Arg394 and the bottom of the channel opened by antagonists. The three MD simulations are all stable, and it can be implied that the complexes generated in the docking calculations are stable.

Discussion In this study, we have investigated the binding of 43 compounds to the hERR LBD by molecular docking simulations. Thirteen structurally diverse setups of the protein were included to investigate the influence of protein conformation on the potential binding mode of the compounds. The docked compounds were chosen to probe likely binding modes of potentially


EDCs and to try to characterize the new conformations obtained from MD simulations (11). Some of these, especially PCBs, have been included in previous modeling studies of the ERR (20, 21, 24). Also, more than 200 compounds have been studied with molecular docking in relation to binding to the androgen receptor (22, 23). These studies include only one or two conformations of the LBD of the nuclear receptor. Initial docking of E2, E3, OHT, and RAL, two endogenous estrogens and two SERMs, into the three X-ray structures of the hERR LBD reproduced ligand binding as observed in the crystal structures with rmsd values for E2 of 0.2-0.3 Å. What cannot easily be deduced from the docking simulations is an appropriate ionization state of His524, which is located in one end of the binding site. On the basis of this observation, docking calculations of all compounds into different protein setups including the three possible charge and tautomer states of this residue are examined. When extending docking of known hERR ligands to also include the four protein structures extracted from MD simulations (11), it is found that these quasi-stable protein structures are diverse from the original X-ray structures and may thus provide the basis for recognition of other small compounds as well as other coregulator peptides. It can be speculated that these conformations are relevant for those identified in peptidebinding studies of the hERR LBD upon interaction with pesticides (19). On the basis of the differences found in the binding of known hERR agonist and antagonist ligands in the structures generated from MD simulations, it can be speculated that these may also be important for other structural studies, for example, in virtual screening and the development of new SERMs. So far, such applications have been based only on structures known from X-ray crystallography, keeping the protein rigid and, inherently, limiting possibilities to find structurally diverse compounds (31-36). Generally, it is difficult for large and flexible ligands to dock in the steroid-binding pocket of the hERR LBD. Examples are FUL, a full hERR antagonist ligand, which is poorly introduced into a few of the included protein structures, and ZEA, which has relatively good experimental binding affinities (26, 56) but cannot be docked properly into the conformations used in this study. For nonylphenol, the problems are especially distinct. Folding of the aliphatic side chain may induce a steroidlike conformation of the ligand, which is not observed here. The poor binding may thus both be due to an unfavorable conformation of the side chain and the absence of a substituent able to mimic the D ring hydroxyl group of E2. Another explanation can be that nonylphenol does not bind in the steroid-binding site but somewhere else on the protein. Other possible binding sites include crevices on the protein surface, for example, the one employed for binding of the coactivator protein (58-61). Genistein and coumestrol, the two phytoestrogens, demonstrate the issue of only employing X-ray structures for docking studies. The two compounds have experimental binding affinities relative to E2 of 5 and 94%, respectively (26). Looking at the overall G-scores, this order is indeed reproduced, but our calculated G-scores for the two phytoestrogens are similar. The relative binding of the two ligands furthermore changes from one structure to another; that is, genistein binds best in the antagonist structures, while coumestrol binds with more favorable G-scores in most of the other setups, which is also what is found structurally for genistein complexed to hERβ LBD (62). Even though the overall binding mode is reproduced, the differences between the binding energies of the two phytoestrogens are thus not easily rationalized on a structural basis.

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Interestingly, steroids and SERMs show a tendency to only bind in specific conformations of the hERR LBD. Other compounds, such as PCBs and pesticides, dock equally in most of the employed hERR LBD conformations, similarly to what has been reported in the literature (20, 21). The position of the ligand in the binding site may change, but the amphiphatic nature of the binding site allows for this without affecting calculated binding affinities considerably. The differences in binding modes and docking scores for PCBs may furthermore show that the PCBs themselves do not bind in the steroid binding site of the hERR LBD, whereas the metabolites bind firmly to this position, hereby competing with E2. The plasticizers included in this study all contain aromatic rings and are highly hydrophobic, whereas the pesticides may be rather hydrophilic, while still containing aromatic rings. Functionally, the pesticides are quite diverse, reflecting the wide differences in the application of different pesticides. G-scores for pesticides (best is HPTE with a G-score of approximately -9 kcal/mol) range from only slightly poorer than for E2 (G score ) -11 kcal/mol) to probably nonbinding (propamocarb with a G-score of ∼-1 kcal/mol) and may depict competitive binding as reflected in the experimental data reported in the literature (26, 27, 38-40). This is especially true under the assumption that pesticides may bioaccumulate and become metabolized, thus increasing the risk of exposure and thereby influencing the conformational equilibrium found in NRs (9, 57). The described trend in this study is that the best binders belong to the class of pesticides, which in a peptide recognition study (19) are proposed to give rise to a new conformation of hERR LBD, which is very intriguing. Furthermore, the observation that these compounds, DDT, its metabolites DDE and HPTE, iprodione, and fenarimol, all have good computed G-scores in all or most of the 13 protein conformations may indicate that they can serve as universal binders to the hERR LBD, regardless of its conformation, hereby effectively competing with the natural ligand. These molecules exhibit binding to hERR LBD where an aromatic part is placed in the steroid cavity in proximity to Glu353 and Arg394. The molecules furthermore extend into the bottom of the channel opened completely by the antagonist ligands, often interacting with Thr347. In conclusion, we have docked multiple compounds including endogenous estrogens, clinically utilized SERMs, and putative EDCs into seven structures of the hERR LBD. The results show the significance of including several protein conformations to better understand the mechanism of EDCs interacting with the LBD. The binding energies calculated in the docking scores are approximate; more comprehensive calculations can be performed for interesting compounds, for example, using the fragment molecular orbital method, which has already been applied to the hERR LBD (63, 64). The method applied here can also be used on other nuclear receptors and proteins in general, assuming structures of multiple conformational states are available and that results can be evaluated with respect to experimental data on binding affinities. The study further reveals that the new quasi-stable conformations can indeed accommodate most of the studied ligands, in many cases with better G-scores, especially the two conformations derived from an apo structure, 7D and 7E, which give rise to the best docking scores computed for many of the ligands across the 13 setups. The availability of novel conformations of NR LBDs is thus of great importance to better comprehend the mechanism of interaction of EDCs and may be of importance for future rational drug design efforts.

Celik et al.

Acknowledgment. We thank the Danish Center for Scientific Computing as well as the Lundbeck, Novo Nordisk, and Carlsberg Foundations for financial support. E. C. B. Jφrgensen is acknowledged for fruitful suggestion.

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