Molecular Basis for the Subtype Discrimination of the Estrogen Receptor-ß-Selective Ligand, Diarylpropionitrile

Jun Sun, Jerome Baudry, John A. Katzenellenbogen and Benita S. Katzenellenbogen

Department of Molecular and Integrative Physiology (J.S., B.S.K.), and Department of Chemistry (J.B., J.A.K.), University of Illinois, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, University of Illinois, Department of Molecular and Integrative Physiology, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}life.uiuc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although the two subtypes of the human estrogen receptor (ER), ER{alpha} and ERß, share only 56% amino acid sequence identity in their ligand binding domain (LBD), the residues that surround the ligand are nearly identical; nevertheless, subtype-selective ligands are known. To understand the molecular basis by which diarylpropionitrile (DPN), an ERß-selective ligand, is able to discriminate between the two ERs, we examined its activity on ER mutants and chimeric constructs generated by DNA shuffling. The N-terminal region of the ERß LBD (through helix 6) appears to be fully responsible for the ERß selectivity of DPN. In fact, a single ER{alpha} point mutation (L384M) was largely sufficient to switch the DPN response of this ER to that of the ERß type, but residues in helix 3 are also important in achieving the full ERß selectivity of DPN. Using molecular modeling, we found an energetically favorable fit for the S-DPN enantiomer in ERß, in which the proximal phenol mimics the A ring of estradiol, and the nitrile engages in stabilizing interactions with residues in the ligand-binding pocket of ERß. Our findings highlight that a limited number of critical interactions of DPN with the ERß ligand-binding pocket underlie its ER subtype-selective character.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS REGULATE MANY important physiological processes, and their targets include reproductive tissues, the cardiovascular system, bone, and the brain (1, 2, 3). The actions of estrogens are mediated through two estrogen receptor (ER) subtypes, ER{alpha} (4) and ERß (5, 6), encoded by different genes. The different tissue distributions of the two ER subtypes and their different effectiveness as transcription regulators suggest that each subtype will have different functions (7, 8, 9).

The two ERs share great homology in their DNA binding domains (97%), but their ligand binding domains (LBDs) share only 56% amino acid sequence identity. Although both subtypes bind to the natural ligand 17ß-estradiol (E2) with high and nearly equal affinity, the differences in their LBDs make the development of ER subtype-selective ligands possible. Such subtype-selective ligands are proving to be useful tools in probing the physiological function of each ER subtype (10) and in studying structure-function relationships of the ligand-receptor complexes. They also have the potential to be useful as estrogen pharmaceutical agents (3, 9, 11).

A number of ER subtype-selective ligands have been developed. A triaryl propyl pyrazole triol was found to be an ER{alpha}-specific agonist, activating gene transcription only through ER{alpha} (12, 13). By adding a basic side chain to the pyrazole core, a highly ER{alpha}-selective antagonist, called methyl piperidino pyrazole, was developed (14). Other compounds, both steroidal and nonsteroidal, that are more potent in activating ER{alpha} than ERß have been described (7, 15, 16, 17, 18, 19, 20, 21). Another class of ER{alpha}-selective ligands are effective agonists on ER{alpha} but are full antagonists on ERß, most notably R,R-diethyl-tetrahydrochrysene (12, 22, 23, 24). Ligands that are ERß selective are also known. Some phytoestrogens such as genistein and coumestrol, as well as some androstanediols, show some selectivity toward ERß, although they activate both ER{alpha} and ERß (7). The ERß selectivity of these compounds, however, does not appear to be good enough for them to be optimal in functional assays.

We have previously identified the nonsteroidal estrogen, diarylpropionitrile (DPN), as an ERß-selective agonist (25). DPN was found to be considerably more potent on ERß than on ER{alpha}. In this study, we have sought to understand the molecular basis for the ERß-selective character of DPN. Toward this goal, we have performed functional assays using mutant ERs generated by site-specific mutagenesis, as well as ER{alpha}/ERß chimeras that could be generated conveniently by DNA shuffling, a novel method for recombination in proteins whose genes have significant nucleotide sequence homology (26, 27, 28, 29, 30, 31, 32). We have also analyzed these results using molecular modeling. We find that one residue in the ligand-binding pocket, Met 336 in ERß, is largely responsible for the ERß selectivity of DPN, but that other residues at the beginning of helix 3 of the LBD are also important contributors to this receptor subtype selectivity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The LBD of ERß vs. ER{alpha} Is the Principal Determining Region for the ERß Selectivity of DPN
We have previously shown that DPN (Fig. 1Go) is an ERß-selective agonist, having higher potency on ERß than on ER{alpha} (25). To determine the extent to which the ERß selectivity of DPN depended on the LBD vs. other receptor domains, we tested DPN on two ER chimeras in which only the LBDs were switched within the context of the remainder of the receptor; these are designated ER{alpha}/ßLBD and ERß/{alpha}LBD (Fig. 2AGo, nos. 2 and 4).



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Figure 1. The Structures of ER Ligands, the Natural ER Ligand E2, and the ERß-Selective Ligand DPN

Note designation of the phenolic A ring of E2 and the {alpha}- and ß-rings of DPN.

 


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Figure 2. The ERß LBD Determines DPN Selectivity, and Met-336 Is the Key Residue

A, Schematic representation of human ER{alpha} and ERß, ER{alpha}-ERß chimeras with LBDs exchanged (ER{alpha}/ßLBD, no. 2; and ERß/{alpha}LBD, no. 4), two receptor point mutants (ER{alpha} L384M, no. 5; and ERß M336L, no. 6), and chimeras in which ER{alpha} sequences corresponding to helices 1–6 or 1–8 in the LBD are introduced into ERß (ERß ({alpha}S395/ßI348, no. 7) and ERß ({alpha}M437/ßL390, no. 8), respectively. Domains A through F and the activation functions (AF-1 and AF-2), DNA binding domains (DBD), and LBDs are indicated. In the ER{alpha}/ßLBD chimera no. 2, the inserted ßLBD residues 255–503 are indicated in parentheses. In the ERß/{alpha}LBD chimera no. 4, the inserted {alpha}LBD residues 302–552 are indicated in parentheses. In the point mutants, the positions of Leu-384 in ER{alpha} and Met-336 in ERß are noted. B, Human endometrial cancer (HEC-1) cells were transfected with expression vectors for wild-type ER{alpha} or ERß or the chimeric ERs no. 2 and no. 4; or panel C, with constructs no. 2, or nos. 4–8, and an (ERE)4-TATA-Luc reporter gene. Cells were treated with the indicated concentrations of DPN for 24 h. Luciferase activity was normalized for ß-galactosidase activity from an internal control plasmid. Values are the mean ± SD for three or more separate experiments and are expressed as a percent of the ER response with 10 nM E2.

 
Unexpectedly, DPN proved to be even more selective on these two chimeras (~100-fold) than on wild-type ERß vs. ER{alpha}, with the selectivity tracking largely with domain E, the LBD (Fig. 2BGo). Transactivation, monitored in human endometrial cancer (HEC-1) cells, by DPN on ERß and ER{alpha}/ßLBD was very similar, but DPN was approximately 3-fold less potent on ERß/{alpha}LBD than on ER{alpha}. Thus, while it is clear that the LBD from ERß is the major factor determining the selectivity of DPN for this ER subtype, other regions from ER{alpha} engender higher DPN potency than those from ERß. This is an important issue, because to properly focus on the molecular factors in the LBD that determine the ER subtype selectivity of DPN, it is essential to make comparisons among ER chimeric constructs that have the same receptor background. In most cases, therefore, we have used chimeras made in the context of ERß (domains A–D and F), because the ERß LBD normally functions in this context.

Met 336 in the ERß LBD Is a Major Factor Determining the ERß Selectivity of DPN
Some 20–25 residues of the ER-LBDs that are in close contact with bound ligands can be identified by analysis of ER-LBD ligand complexes (33, 34). Of these, all but two are identical in ER{alpha} and ERß, and these two differences are conservative: Leu-384 and Met-421 in ER{alpha} vs. Met-336 and Ile-373 in ERß, respectively.

Initially, several point mutants were made in the LBD, and these receptors were tested for their transactivation by DPN. Of the mutants examined, the only one that had a major effect on the ER subtype selectivity was that in which the corresponding residues, Leu-384 in ER{alpha} and Met-336 in ERß, were exchanged (Fig. 2CGo). The dose-response curve for DPN on the ER{alpha} L384M mutant (Fig. 2AGo, no. 5) shows a pronounced left shift, compared with that for ER{alpha}, toward that observed with the ER{alpha}/ßLBD. Thus, simply by changing Leu-384 in the ER{alpha} LBD pocket into the ERß type residue Met, we were able to shift the selectivity of DPN to that characteristic of the ERß type. Significantly, by mutating Met-336 in ERß to the ER{alpha} residue Leu (Fig. 2AGo, no. 6), we shifted the dose-response curve for DPN to the right, toward that observed with the ERß/{alpha}LBD (Fig. 2CGo).

Although these single amino acid substitutions resulted in major shifts in the dose-response curves of DPN, the selectivity of DPN on ER{alpha} L384M and ERß M336L was still approximately 3-fold less than it was on the appropriate comparator receptors, ERß/{alpha}LBD and ER{alpha}/ßLBD, respectively. Thus, other residue(s) in the LBDs must contribute to the ERß selectivity of DPN, although clearly, Met-336 in ERß is a major factor.

The N-Terminal Region of the ERß LBD (through Helix 6) Is Fully Responsible for the ERß Selectivity of DPN
To investigate the role of region(s) in the ERß LBD, other than the amino acid Met 336, in determining DPN ERß selectivity, we tested two ERß chimeras generated by cloning, containing hybrid LBDs [ERß ({alpha}S395/ßI348), chimera no. 7, and ERß ({alpha}M437/ßL390), chimera no. 8]. These chimeras, which incorporate ER{alpha} LBD sequences through helix 6 or through helix 8 (see Fig. 2AGo), respectively, also cover one (Leu-384 or Met-336 in ER{alpha} and ERß, respectively), or both of the different residues in the binding pocket (Leu-384 and Met-421 in ER{alpha} vs. Met-336 and Ile-373 in ERß), respectively (Fig. 2AGo). A simple nomenclature system has been developed to describe the chimeras we have studied; the crossover sites from ER{alpha} LBD to ERß LBD sequences or the reverse are designated as ({alpha}X###/ßY###) or (ßY###/{alpha}X###), respectively.

DPN had the same character on both chimeras nos. 7 and 8, which was also the same as it had on ERß/{alpha}LBD (Fig. 2CGo). Therefore, by replacing the N-terminal region of the ERß LBD (helix 1 through helix 6) with its counterpart from ER{alpha}, we were able to switch the character of the response to DPN to the ER{alpha} type. Thus, in addition to Met-336, other residue(s) in this region play some role in determining DPN selectivity.

Generation of ERß-ER{alpha} Chimeras by DNA Shuffling for Further Analysis of the ERß Selectivity of DPN
To make a more global analysis of the ER-LBD sequences that are responsible for the subtype selectivity of DPN, we used DNA shuffling between the LBDs of the two ER subtypes to generate numerous chimeras within the N-terminal region (helix 1–6) of these LBDs. The general scheme used to produce these chimeras by DNA shuffling is outlined in Fig. 3Go.



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Figure 3. Schematic of the Methodology Used for DNA Shuffling of the ER{alpha} and ERß LBDs, and Generation of the ER{alpha}-ERß LBD Chimeras Used to Investigate the Selective Agonistic Character of DPN on ERß

 
In brief, the shuffling is done by a multistep process that consists of 1) mixing ER{alpha} and ERß LBD cDNA sequences; 2) limited digestion with deoxyribonuclease I to produce appropriately sized fragments; 3) PCR without addition of oligonucleotide primers, to assemble longer cDNA LBD fragments that contain portions from the two genes, with crossovers occurring where there is sufficient sequence homology for cross-priming; 4) PCR with primers that correspond to the desired N and C termini from the ER subtype LBD cDNAs; and 5) treatment of the appropriately sized chimeric LBD cDNA amplicon with SacII and MfeI followed by insertion into the SacII/MfeI digested pCMV5-ERß vector, replacing the corresponding wild-type ERß LBD fragment.

Because N-terminal sequences in the ERß LBD through helix 6 seemed to account fully for the ERß selectivity of DPN, we focused our attention only on chimeras with crossovers in this region. As indicated by the highlights in Fig. 4AGo, seven sequence regions in which crossovers occur were found within this area, and analysis of the functional activity of these chimeras was undertaken. The ERß chimeras generated have mostly ERß sequence, with mosaic sequences within the N-terminal portion of the LBD (Fig. 4BGo). As was discussed above, these mosaics were studied in a single ER subtype context, that of ERß, so as to monitor effects in a fixed receptor background in which only the LBDs were altered. The direction of the crossovers (i.e. ER{alpha} to ERß or ERß to ER{alpha}) in the various chimeric sequences was determined by the primers that were used in the second PCR step in the DNA shuffling process, and these are indicated by the arrows shown at the crossover sites (Fig. 4AGo). As expected, the crossovers occur only in regions of high homology between the two sequences.



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Figure 4. Crossover Sites and ER{alpha}/ERß Chimeras Generated by DNA Shuffling

A, The alignments of DNA and protein sequences between human ER{alpha} and ERß at the N-terminal part of the LBD. The crossover sites for the ERß chimeras generated from DNA shuffling are highlighted. The chimeric sequence was inserted into the SacII/MfeI sites of pCMV5-ERß. The orientation of the chimeric sequence is indicated by the arrows at the crossover sites. The helix structure of the LBD is diagrammed at the top, and is according to the genistein-ERß structure (33 ). B, Schematic representations of the chimeras. Each chimeric construct is given a number.

 
Helix 3 of ERß Is Also Involved in Determining DPN Selectivity for this ER Subtype
The transactivation by DPN on one chimeric ERß (ßE274/{alpha}A322) (no. 12) (see Fig. 4BGo) was found to be identical to that of ERß/{alpha}LBD, indicating that the region between the end of helix 2 to helix 6 is responsible for the reduced potency of DPN on ER{alpha} (Fig. 5Go). By contrast, another chimeric ERß (ßI316/{alpha}P365; no. 15) (see Fig. 4BGo), which has a longer N-terminal sequence from ERß with the crossover site in helix 4, had a character very similar to ERß M336L (Fig. 5Go). This suggests that Met-336 is the only important residue within the helices 4–6 region and that the other site(s) important to establish the full potency of DPN on ERß must reside within helix 3, where the core of the LBD starts.



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Figure 5. Helix 3 of the ERß LBD Is Also Important for DPN Selectivity

Transfection/transactivation assays were conducted in HEC-1 cells using some selected ER constructs, as described in the legend for Fig. 2Go.

 
Several ERß chimeras have crossover sites in helix 3, with N-terminal sequences from ER{alpha} and C-terminal sequences from ERß. DPN was less potent on these ERß chimeras compared with wild-type ERß (Fig. 6AGo). One of the constructs, ERß ({alpha}N348/ßL301; no. 9) (see Fig. 4BGo), has only three amino acid residues different from ERß at the beginning of helix 3. DPN lost approximately 5-fold of its potency selectivity on this chimera. This was intriguing because these three residues at the start of helix 3 are not considered to be in contact with the ligand. Molecular modeling, presented below, suggests a way in which these three residues might be affecting ligand binding.



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Figure 6. The N-Terminal Residues in Helix 3 from ERß Contribute to DPN Selectivity

Transfection/transactivation assays were conducted in HEC-1 cells using some selected ER constructs, as described in the legend for Fig. 2Go. In panel A, the ER constructs used have a mosaic LBD with N-terminal sequence from ER{alpha}. In panel B, the ER constructs used have N-terminal sequence from ERß.

 
Two other chimeras (nos. 13 and 15, see Fig. 4BGo) also have crossovers in helix 3, but instead have ERß N-terminal sequences and ER{alpha} C-terminal sequences. The data from these chimeras also suggest that the amino acid residues at the beginning of helix 3 are important for DPN selectivity (Fig. 6BGo). For example, chimera nos. 13 and 15 (and no. 14, data not shown), which include a few of the ERß helix 3 residues that are different from ER{alpha}, show increased DPN potency compared with ERß/{alpha}LBD. Taken together, it appears that three ERß residues at the beginning of helix 3, Met-296, Ser-297, and Lys-300, are also important for the maximal efficacy and selectivity of DPN on ERß.

Investigation of the Modes of DPN Binding to the ER Subtypes by Molecular Modeling
DPN is a chiral molecule and exists as R and S enantiomers. So far, however, attempts to separate these enantiomers or prepare them by an enantio-selective synthesis have been unsuccessful (Treutle, M., and J. A. Katzenellenbogen, unpublished). Therefore, the ligand used in these biological studies is a racemate, consisting of both S- and R-DPN. To model the interactions of DPN with the ER subtypes, both of the DPN enantiomers were docked separately into the LBDs of ER{alpha} and ERß. Also, because it is not certain which of the two aryl rings of DPN is playing the role of the A ring of E2 in the ligand-binding pocket, we examined models in which the {alpha}-ring and the ß-ring were separately initially positioned at the A ring binding pocket (see Fig. 1Go). These models were developed concurrently with our experimental studies. In each case, we examined a number of alternative starting geometries and then subjected the docked structures to rigorous energy minimization to obtain final, stable structures (see Materials and Methods for details).

Methionine-336 in ERß as a Determinant of DPN Selectivity
For S-DPN, the most stable structure we obtained from modeling this ligand in the LBDs of either ER{alpha} or ERß (Fig. 7AGo) is one in which the ß-ring of S-DPN is positioned at the A ring binding pocket (cf. Fig. 1Go). The p-OH on the ß-ring forms good hydrogen bonds with ER{alpha} Glu-353 and Arg-394 (or ERß Glu-305 and Arg-346), and the p-OH on the {alpha}-ring forms a hydrogen bond with ER{alpha} His-524 (or ERß His-475) at the other end of the cavity. These are the very same hydrogen bonds found in the ER complexes with E2 and other high-affinity ligands, such as diethylstilbestrol and raloxifene (22, 34). In this orientation, the nitrile group of S-DPN projects into the ß-face of the cavity, making close contact (i.e. within 4.2 Å) with a series of receptor residues (ER{alpha} Ala-350, Trp-383, Leu-384, Leu-387; or ERß Thr-299, Ala-302, Trp-335, Met-336, Val-487) (Figs. 7AGo and 8Go). In fully minimized structures, S-DPN has significantly stronger interaction energy with ERß than with ER{alpha}.



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Figure 7. Crossed Stereo View of S-DPN (Panel A) and R-DPN (Panel B) Docked and Minimized in the ER{alpha} and ERß LBD Pockets, Respectively

DPN and the ERß pocket residues are shown with standard atom colors, whereas in the ER{alpha} complex, DPN and the pocket residues are shown in orange. Three hydrogen bonding residues (Glu, Arg, and His) and one of the different residues in the pocket (ER{alpha} M421 vs. ERß I373) are shown, as well as some side chains from selected residues that are close to the nitrile group. The backbones of the two ERs are superposed based on their main chain atoms from helix 3 to helix 11. The figure was generated in SYBYL.

 


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Figure 8. Crossed Stereo View of S-DPN in the ER{alpha} and ERß LBD Pockets, Showing Contact with Helix 3

DPN and the ERß pocket residues are shown in standard atom colors, whereas in the ER{alpha} complex, DPN and the pocket residues are shown in orange. The backbone of the two ERs are superposed based on their C{alpha} atoms. Only the first half of helix 3 is shown, and selected side chains have been deleted for clarity. This view is taken from the same structures as shown in Fig. 7Go. The figure was generated in SYBYL.

 
The distance between the nitrile of S-DPN and Met-336 in ERß or Leu-384 in ER{alpha} is particularly close, about 3.6–3.8 Å. This is an interesting finding, because of the role that this residue was found to play in determining the ERß selectivity of DPN, as was established by the preceding mutagenesis studies. Thus, we believe that a more favorable interaction occurs between the nitrile group and ERß Met-336 than between ER{alpha} Leu-384, and that this is a significant factor determining the ERß-selective character of DPN, although other more global factors also contribute.

Because the nitrile and the methionine sulfide both have electron lone pairs, it is not at first obvious that a favorable interaction could develop between them. The geometry of interaction is important here: it is not directly end on, with the sulfur in line with the carbon-nitrogen triple bond; rather, it is side on, with the sulfur approaching the side of the nitrile group near the carbon atom at an angle of 112° where energetically favorable interactions could take place between donor molecular orbitals on sulfur and acceptor molecular orbitals on the nitrile (Fig. 7AGo).

Curiously, the most favorable fit for R-DPN into the binding pocket of the ER LBDs is different in the two subtypes. In ERß, its ß-ring is preferred as the A ring mimic, whereas in ER{alpha}, the {alpha}-ring is preferred (Fig. 7BGo). In both cases, the nitrile group of this enantiomer projects into the {alpha}-face of the cavity (opposite to that of S-DPN). The only polar residue in this region of the ER subtypes is Met-421 in ER{alpha}, and it is too far from the nitrile and at a poor geometry for any productive interaction. In fully minimized structures, R-DPN has a more favorable interaction energy with ER{alpha} than with ERß. It is noteworthy, however, that in absolute terms, R-DPN has a weaker interaction for its preferred subtype (ER{alpha}) than does S-DPN for its preferred subtype (ERß), although the interaction of R-DPN with ER{alpha} is stronger than is the interaction of S-DPN for ER{alpha}. Thus, it appears that the ERß-selective character of the DPN racemate is the result of the dominance of the more strongly interacting, ERß-selective enantiomer (S-DPN) over the more weakly interacting, ER{alpha}-selective enantiomer (R-DPN).

Differences in Interaction with a Threonine in Helix-3 Appear to Support DPN ERß Selectivity
In wild-type ERß, the nitrile of S-DPN is relatively close to Thr-299, at the start of helix-3, so that a hydrogen bond between these groups might form. There is a threonine residue in ER{alpha} (Thr-347) that corresponds to Thr-299 in ERß; however, in the S-DPN-ER{alpha} LBD complex, the position of the ligand is such that the nitrile is considerably further from Thr-347; therefore, formation of a hydrogen bond is unlikely. The difference in nitrile-threonine distances is particularly clear in the view shown in Fig. 8Go. Thus, this threonine residue appears to be a site that might also contribute to DPN selectivity, because although this residue is conserved between the two ER subtypes, only in S-DPN in the ERß complex is the nitrile close enough to make a hydrogen bond feasible (Figs. 7AGo and 8Go).

The fact that a hydrogen bond might form between the nitrile and Thr-299 in ERß, whereas the greater ligand-residue separation clearly precludes formation of the corresponding nitrile-Thr-347 hydrogen bond in ER{alpha}, suggests that differences between ERß and ER{alpha} at the start of helix 3, which is near this threonine, might also be playing a role in the ERß subtype selectivity of DPN. Surrounding this threonine at the start of helix 3 are residues, three of which differ very significantly between ER{alpha} and ERß (Met-296, Ser-297, and Lys-300 surround Thr-299 in ERß, whereas Gly-344, Leu-345, and Asn-348 surround Thr-347 in ER{alpha}).

Although they are not in direct contact with DPN, these sequence differences appear to perturb the overall ER structure in a way that affects the interaction of the DPN nitrile with the nearby Thr residue. In fact, when the portion of the LBDs around the DPN ligand are superposed, a significant shift can be noted between the backbones of the two helix 3’s, as well as between the precise position of the two ligands (Fig. 8Go). The overall effect of this is that the ligand-binding pocket near the start of helix 3 in ERß is narrower than in ER{alpha} and that S-DPN makes a tighter, more comfortable fit into the narrower pocket of ERß; this brings the nitrile closer to Thr-299, engendering hydrogen bond formation. (Note in Fig. 8Go that helix 3 is closer to DPN in the ERß structure than in the ER{alpha} structure.) This finding from modeling matches well with our observations in the functional assays of ER{alpha}-ERß LBD chimeras that these same three residues at the start of helix 3 also make a contribution to the ERß selectivity of DPN.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ERß Subtype Selectivity of DPN Is Determined by Relatively Few Residues in the Ligand-Binding Pocket
We have used a combination of functional analysis of site-specific mutants and ER{alpha}-ERß LBD chimeras generated by DNA shuffling, coupled with molecular modeling, to characterize the molecular basis for the ERß selectivity of the nonsteroidal ligand, DPN. We find that by changing one relatively conserved residue in the ligand-binding pocket between ER{alpha} and ERß (L384 in ER{alpha} and M336 in ERß), we can reverse, to a large extent, the ERß selectivity of DPN, although changes in helix 3 are needed to achieve a full shift in ER subtype selectivity. Through molecular modeling, we have found that the change at the ER{alpha} L384/ERß M336 site in the LBD alters not only the local interaction with ligand, but also seems to have a long-range influence on other parts of the binding pocket. This matches well with the results we have obtained from the functional assays.

DNA Shuffling Provides a Rapid Way to Generate Multiple ER{alpha}-ERß Chimeras Needed for Functional Studies
In our investigations of the molecular basis for the ERß subtype selectivity of DPN, we have applied the novel method of DNA shuffling to generate rapidly many chimeric ER LBDs (26, 27, 28, 29, 30, 31, 32). Chimeras have played an important role in evaluating the function of nuclear hormone receptors, beginning with the delineation of the respective roles of the DNA- and ligand-binding domains (8, 35, 36) and the relative effectiveness of the N- and C-terminal activation functions, AF-1 and AF-2 (37, 38). By contrast, analysis of the function of a single domain, such as the LBD, has typically relied more often on site-specific mutational substitution and truncation than on generation of chimeras (39, 40, 41, 42).

Intradomain chimeras, where they have been used, have typically been generated by labor-intensive cloning methods. DNA shuffling (26, 27, 28, 29, 30, 31, 32) provides an alternative method for generating chimeric structures that is rapid and convenient. However, this method requires that the genes to be shuffled have sufficient nucleotide sequence homology that cross-priming will take place in the first rounds of PCR. The ER{alpha} and ERß LBDs are close to the limit in nucleotide sequence divergence for shuffling to be successful (63% nucleotide sequence identity).

The crossovers in chimeras generated by DNA shuffling are not distributed uniformly throughout the domain, but are concentrated in regions where nucleotide sequence homology is highest. Also, the only control one has over the distribution of crossovers in shuffling-generated chimeras comes through the selection of the oligonucleotide primers used in the second PCR reaction. Thus, using 5'-{alpha}/3'-ß or 5'-ß/3'{alpha} primer sets, one can ensure that the N and C termini of a chimera will be {alpha}/ß or ß/{alpha}, respectively. The number of crossovers in such primer sets will be odd, and typically will be one. When the 5'- and 3'-primers are both sequences from the same ER subtype, it is most likely that wild-type, unshuffled sequences will be obtained, although some chimeras with an even number of crossovers will also be found.

Analysis of the Molecular Basis of Ligand Selectivity in ER and Other Systems
Hormones for nuclear receptors are typically very selective, although the molecular basis for this selectivity has been studied in only a limited number of systems. As observed with DPN, where only a few residues were found to determine receptor ER subtype selectivity, the species selectivity of the antiprogestin RU38,486 between chick and human progesterone receptor (PR) was found, by analysis of interspecies chimeric PRs, to depend on a single residue, which in human PR was a glycine and in chick PR was a cysteine (43). The large 11ß substituent on RU38,486 could be accommodated by the larger pocket in human PR, but clashed with the larger residue at this site in chick PR. In attempts to find the basis for the hormonal discrimination between the PR and the androgen receptor (AR), by investigating the activity of progestins and androgens on various chimeric constructs generated by cloning, however, hormonal discrimination between progestins and androgens appeared to depend on multiple sequences rather than only a few residues (44).

In an earlier study from our laboratories, completed before the crystal structure of the ER{alpha} LBD was solved, we used site-specific mutagenesis as a follow up to an analysis of ligand binding through alanine scanning mutagenesis, to ascertain what residue in ER{alpha} was the hydrogen-bonding partner of the A ring phenol in E2 (40, 45). Critical to this study was a comparison between the LBD sequences of ER{alpha} and AR in the region thought to be in close proximity to this phenol and the corresponding keto function in AR ligands. By substituting single AR residues into ER, we were able to determine that Glu-353 in ER{alpha} was an important hydrogen-bonding partner of the phenol, and that the Gln residue in the corresponding position of AR was the partner for the androgen C-3 ketone (41), results later confirmed by x-ray crystallography (34, 46).

Other studies on nuclear hormone receptor subtype selectivity, in the peroxisome proliferator-activated receptors (47, 48) and the retinoic acid and the retinoid X receptors (49), have involved molecular modeling and site-specific mutagenesis approaches guided by x-ray crystal structures of ligand-receptor complexes. In most cases, the subtype selectivity of ligands was found to depend on only a few differences in ligand-residue contacts within the ligand-binding pocket.

The results we have obtained in this study on ligand selectivity between ER{alpha} and ERß subtypes point to a discrimination that is also based on a relatively few number of interactions, as was the case with the specificity of RU 486 on PRs from different species (43) and with ligands for the peroxisome proliferator-activated receptors, retinoic acid receptors, and retinoid X receptors (47, 48, 49). What is notable in our findings, however, is that some of these interactions appear to be modulated by sequence differences that are somewhat more distant from the residues that are in direct contact with the ligand.

Molecular Modeling Suggests that the S-Enantiomer of DPN Is Responsible for the ERß Selectivity of the Racemate
Although there are, in principle, eight different complexes that can form between the ER LBD and DPN, four for each enantiomer, a number of these were eliminated from consideration as a consequence of our molecular mechanics-based modeling. An energetically and structurally satisfying fit was found with the S-enantiomer of DPN, in which the ß-ring of DPN functions as the mimic of the phenolic A ring of E2 and the nitrile projects toward the ß-face of the ligand-binding pocket. In this orientation, the nitrile is positioned where it might engage in energetically productive interactions with Met-336 and Thr-299 in ERß, but the corresponding interactions in ER{alpha} (with Leu-384 and Thr-347) are not possible.

The differential interaction of the nitrile with Met-336 in ERß vs. Leu-384 in ER{alpha} appears to be simply a consequence of residue differences, but the potential interaction of the nitrile with Thr-299 in ERß vs. its lack of possible interaction with Thr-347 in ER{alpha} seems to result from sequence differences at the start of helix 3 that are rather remote from the ligand and that reshape the ligand-binding pocket, making it smaller in ERß than in ER{alpha}. In these models, S-DPN, being a small ligand, fits more snugly in the ERß ligand pocket, in a manner that positions its nitrile rather close to Thr-299, whereas the larger pocket in ER{alpha} does not engender such a nitrile-threonine approximation.

This satisfying model notwithstanding, one should note that the computer-generated models we have investigated represent the most stable structures that we can obtain by simple molecular mechanics modeling, and they originate from x-ray crystal structures of complexes with different ligands. Thus, while one would expect that only one of the several possible orientations would have the lowest energy level, during our modeling process, we found that the energy differences between related complexes were often small. It will certainly be interesting to see whether a single or multiple orientations for a certain enantiomer of DPN are found when the crystal structure of the DPN-liganded ERß LBD is solved. There is already one example of multiple ligand orientations found in the x-ray crystal structure of a complex with a nuclear hormone receptor, the progestin X receptor/steroid X receptor and a bisphosphonate ligand (50); however, in that case, the ligand-binding pocket is much larger and more open than is the case with the ERs. Nevertheless, based on the modeling and the functional studies that we have done, we propose that S-DPN would be a higher affinity and more ERß-selective compound than R-DPN, and that enantiomerically pure S-DPN would be more ERß selective than the DPN racemate. It is hoped that challenges in the preparation of the individual DPN enantiomers can be overcome, so that our prediction that S-DPN is the more selective enantiomer can be verified experimentally.

Our findings highlight that a limited number of critical interactions of DPN with the ERß ligand-binding pocket underlie its ER subtype-selective character. In addition, this study illustrates the utility of DNA shuffling methodology for the rapid generation of receptor chimeras that should be broadly applicable to the functional analysis of other nuclear hormone receptors that are derived from different genes with significant nucleotide sequence homology.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals, Materials, and Plasmid Constructions
Cell culture media were purchased from Life Technologies, Inc. (Gaithersburg, MD). Calf serum was obtained from HyClone Laboratories, Inc. (Logan, UT), and fetal calf serum was purchased from Atlanta Biologicals (Atlanta, GA). The luciferase assay system was from Promega Corp. (Madison, WI). The ERß-selective ligand studied in this report, DPN, was prepared as described (25).

The expression vector for human ER{alpha} (pCMV5-hER{alpha}) was constructed previously as described (39). The expression vector pCMV5-ERß was constructed by inserting the cDNA encoding the human ERß (8) into the BamHI site of pCMV5. ER point mutants were generated by QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA).

A number of chimeras of ER{alpha} and ERß have been constructed from portions of the ER{alpha} and ERß LBDs, and these are designated by the following nomenclature: in each case the crossover point is designated as ({alpha}X###/ßY###) or (ßY###/{alpha}X###), where the X###’s and Y###’s specify the residue positions in the ER{alpha} or ERß LBDs, respectively, at the site of the crossover; the sequence of the ER outside of the LBD is specified as being of the {alpha}-type by ER{alpha} and of the ß-type as ERß.

Two ERß chimeras, ERß ({alpha}S395/ßI348) and ERß ({alpha}M437/ßL390), were constructed by first changing the codons for ERß P253 and R254 into CCGCGG, which generates a SacII site, and then inserting a PCR-generated ER{alpha} fragment into the pCMV5-ERß vector, replacing SacII/MfeI and SacII/AflII fragments, respectively. As for the pCMV5-ERß expression vectors, which have chimeric sequences before the end of helix 6, they were generated by inserting PCR-amplified chimeric sequence from the DNA shuffling products (preparation described below) into the SacII/MfeI sites, replacing the wild-type sequence.

The estrogen-responsive reporter plasmid was (ERE)4-TATA-Luc, kindly provided by Dr. David Shapiro (University of Illinois). The plasmid pCMVß (CLONTECH Laboratories, Inc., Palo Alto, CA), which contains the ß-galactosidase gene, was used as an internal control for transfection efficiency.

DNA Shuffling
DNA shuffling was performed by a modified method we have recently described (51). Primers ER{alpha} A289f (5'-GCCAACCTTTGGCCAAGCCC-3') and ER{alpha} S566r (5-GCTTTGGTCCGTCTCCTCCAC-3') were used to amplify ER{alpha} LBD cDNA from a plasmid template. To amplify ERß LBD cDNA, primers ERß A242f (5'-GCCGGCAAGGCCAAGAGAAGT-3') and ERß E516r (5'-CTCTGCCGGGCTGCACTC-3') were used. After gel purification, about 2 µg of ER{alpha} LBD and ERß LBD cDNAs were mixed, and 2.5 µl of 1 M Tris-HCl (pH 7.5) and 2.5 µl of 200 mM MnCl2 were added. The volume was brought to 49 µl with distilled H2O, and the mixture was equilibrated at 15 C for 5 min. Deoxyribonuclease I (1 µl of 10 U/µl, Life Technologies, Inc.) was freshly diluted 1:100 in distilled H2O, and digestion was performed at 15 C for 90 sec. The reaction was stopped by adding 5 µl of ice-cold stop buffer containing 50 mM EDTA and 30% (vol/vol) glycerol. The digested products were separated by electrophoresis in a 2% (wt/vol) agarose gel, and DNA fragments of about 100 bp were recovered from the gel. About 0.5 µg of product was combined with 0.5 µl of Pfu polymerase, 5 µl of 10x Pfu polymerase buffer, 2 µl of 2.5 mM deoxynucleoside triphosphate in a total volume of 50 µl. The reassembling reaction was performed in a PCR machine (PTC-100, MJ Research, Inc., Watertown, MA) under the following conditions: 60 sec at 94 C followed by 30 cycles of 30 sec at 94 C, 1 min at 40 C, and 1 min at 72 C. The recombined products were amplified in a standard PCR, using 1 µl of 1:10 diluted reassembling products as template, ERß EfSacII (5'-CACGCGCCGCGGGTGCGGGAGCTGCTGCTG-3') and ER{alpha} H6rMfeI (5'-CTCAATTGAGCGCCAGACGAGA-3') or ER{alpha} EfSacII (5'-ATCAAACCGCGGAAGAAGAACAGCCTGGCC-3') and ERß H6rMfeI (5'-GTCAATTGAGCGCCACATCAGC-3') as primers. After gel purification, the proper sized DNA amplification products were digested with SacII/MfeI and inserted into SacII/MfeI-digested pCMV5-ERß vector fragment, as described above (the codons for ERß P253 and R254 were changed to CCGCGG, which generates a SacII site). The sequences of all chimeric constructs were checked by sequencing.

Cell Culture and Transient Transfections
Human endometrial cancer (HEC-1) cells were maintained in culture as described (12). Transfection of HEC-1 cells in 24-well plates used a mixture of 0.35 ml of serum-free improved MEM and 0.15 ml of Hanks’ Balanced Salt Solution containing 5 µl of lipofectin (Life Technologies, Inc.), 1.6 µg of transferrin (Sigma, St. Louis, MO), 0.5 µg of pCMVß as internal control, 1 µg of the reporter gene plasmid, 100 ng of ER expression vector, and carrier DNA to a total of 3 µg DNA per well. The cells were incubated for 8 h at 37 C in an incubator containing 5% CO2. The medium was then replaced with fresh medium containing the desired concentrations of ligand. Reporter gene activity was assayed at 24 h after ligand addition. Luciferase activity, normalized for the internal control ß-galactosidase activity, was assayed as described (12).

Molecular Modeling
The genistein-ERß LBD crystal structure [Protein Data Bank code 1QKM (33)] was imported into SYBYL (Tripos Inc., St. Louis, MO) or MOE (Molecular Operating Environment, Chemical Computing Group Inc., Montréal, Québec, Canada). After hydrogen was added and heavy atoms were fixed, energy minimization was performed using the MMFF94 force field. The same procedure was applied to the diethylstilbestrol-ER{alpha} LBD crystal structure [Protein Data Bank code 3ERD (22)]. Both enantiomers of DPN, R-DPN and S-DPN, were built in SYBYL and in MOE, using the MMFF94 force field, and the lowest-energy conformers of each enantiomer were then docked in various orientations into the ERß and ER{alpha} LBD. Docking and minimization were done using routines previously described (13), and details can be found in that reference. Separate dockings were done in which either the {alpha}-ring or the ß-ring of both R- and S-DPN were initially placed into the A ring-binding pocket. The energetically most favorably docked ligand-LBD structures were then further minimized, as described previously (13). Interaction energies were evaluated after this final minimization by calculating the difference between the energy of the ER-ligand complex and the sum of the energies of the ligand and ER alone.

The structures of the ER{alpha} LBD mutant L384M and the ERß LBD mutant M336L were generated in SYBYL and MOE by mutating residues in the templates prepared as above and energy minimization of the altered site chains. Both R-DPN and S-DPN were modeled into the mutant receptors as above.


    ACKNOWLEDGMENTS
 
We thank Dr. Huimin Zhao for advice on DNA shuffling and Alice L. Rodriguez for assistance with molecular modeling.


    FOOTNOTES
 
This work was supported by grants from the NIH [PHS 5R01 CA-18119 (to B.S.K.) and PHS 5R37 DK-15556 (to J.A.K.)].

Abbreviations: AR, Androgen receptor; DPN, diarylpropionitrile; E2, 17ß-estradiol; ER, estrogen receptor; LBD, ligand binding domain; Luc, luciferase; PR, progesterone receptor.

Received for publication September 30, 2002. Accepted for publication November 15, 2002.


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