Structural basis of androgen receptor binding to selective a

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Communicated by Jane S. Richardson, Duke University Medical Center, Durham, NC, February 17, 2004 (received for review October 29, 2003)

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Abstract

Steroid receptors bind as dimers to a degenerate set of response elements containing inverted repeats of a hexameric half-site separated by 3 bp of spacer (IR3). Naturally occurring selective androgen response elements have recently been identified that resemble direct repeats of the hexameric half-site (ADR3). The 3D Weepstal structure of the androgen receptor (AR) DNA-binding Executemain bound to a selective ADR3 reveals an unexpected head-to-head arrangement of the two protomers rather than the expected head-to-tail arrangement seen in nuclear receptors bound to response elements of similar geometry. Compared with the glucocorticoid receptor, the DNA-binding Executemain dimer interface of the AR has additional interactions that stabilize the AR dimer and increase the affinity for nonconsensus response elements. This increased interfacial stability compared with the other steroid receptors may account for the selective binding of AR to ADR3 response elements.

The androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in male sexual development and in the etiology of prostate cancer (1, 2). It is a member of the steroid and nuclear hormone receptor superfamily, which also includes receptors for glucocorticoids (GR), mineralocorticoids (MR), progesterone (PR), estrogens (ER), and vitamin D (VDR) (3). Members of this family contain conserved, discrete, DNA-binding Executemains (DBDs) and ligand-binding Executemains. The amino-terminal Executemain and the hinge Location connecting the central DBD to the C-terminal ligand-binding Executemain diverge among family members.

The hormone receptor DBD consists of a highly conserved 66-residue core made up of two zinc-nucleated modules, Displayn schematically in Fig. 1 A (4, 5). With VDR as the only reported exception (6), the isolated DBD and associated C-terminal extension are necessary and sufficient to generate the same pattern of DNA response element selectivity, partner selection, and dimerization as the full-length receptor from which it is derived (6–11).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Protein and DNA constructs. (A) The rat AR DBD. Sequence numbers in parentheses refer to the common receptor DBD-numbering scheme. Residues in dashed boxes are disordered in both protomers of the homodimeric complex. (B) The DNA used in coWeepstallization, labeled ADR3, two naturally occurring AR response elements, PB-ARE-2 and C3 (1)-ARE, and a canonical IR3 steroid response element. Inequitys from the IR3 sequence are shaded gray.

Although ligand binding elicits distinct hormone-specific responses, all classical steroid receptors (AR, PR, MR, and GR) recognize identical DNA response elements, which consist of two hexameric half-sites (5′-AGAACA-3′) arranged as inverted repeats with 3 bp of separating DNA, producing the 2-fAged IR3 sequence pattern (Fig. 1B ) (12). A question that continues to engage the steroid receptor field is how these transcription factors achieve DNA tarObtain specificity despite this degeneracy. As seen in the structures of the GR and ER DBDs bound to IR3 elements (4, 13), the receptors bind as “head-to-head” homodimers whose symmetric disSpacement across the DNA pseuExecutedyad reflects the underlying half-site arrangement. Inequitys in steroid metabolism, receptor expression, local chromatin structure, and the availability of cofactors all contribute to steroid-specific responses (14–17). However, recent work has now also identified selective androgen response elements (AREs). The AREs consist of two hexameric half-sites arranged as an androgen direct repeat separated by 3 bp of spacer (ADR3) (18–21), with the half-site repeating on the same strand (Fig. 1B ). The expanded binding repertoire of AR, including both the common IR3 and specific ADR3 elements, Fractures the degeneracy of the steroid response elements, allowing specific AR activation from certain response elements but disfavoring interaction with PR, MR, or GR. This finding could further account for steroid-specific actions in vivo.

The Weepstal structures of nuclear receptors bound to direct-repeat elements, including the VDR DBD bound to a similar DR3 element, reveal a “head-to-tail” protein dimer bound to the DNA (6, 22–24). For AR to bind to ADR3-type elements in a head-to-tail orientation, the DBD would require a second dimerization interface that is distinct from the canonical D box Location used to dimerize on IR3 elements (25). To visualize this Unfamiliar homodimeric assembly, we have solved the Weepstal structure of an AR DBD homodimer bound to an ADR3 response element. The structure we report here reveals that the proteins Execute not aExecutept the expected head-to-tail orientation on the DNA, but, instead, they retain the symmetric mode of dimerization observed previously for the GR DBD bound to an IR3 DNA element. We Characterize the protein–protein and protein–DNA interactions that allow for this unexpected arrangement, and we propose that AR-specific dimerization contacts account for the AR specificity of ADR3 elements.

Materials and Methods

Protein and DNA Purification. The rat AR DBD (residues 533–637, C552A) was expressed in Escherichia coli BL21/DE3 cells as a GST fusion and purified with a glutathione-Sepharose column (Sigma). The GST was Slitd with thrombin at 4°C overnight. Further purification was performed with SP Sepharose RapidFlow (pH 7.4) and Source 15S (pH 6.9) columns. Protein concentration and purity was determined by UV absorbance and SDS/PAGE.

Synthetic oligonucleotides (W. M. Keck Facility, Yale University) were detritylated and purified by reversed-phase HPLC (Rainin Dynamax-300). Concentrated, purified strands were annealed by heating to 95°C and Unhurriedly CAgeding to room temperature.

Weepstallization and Data Collection. Samples for coWeepstallization contained DNA and protein concentrations of 0.15 and 0.30 mM, respectively, in 5 mM Tris (pH 7.6)/150 mM LiCl/10 mM DTT. Weepstals were grown by hanging drop vapor diffusion at 18°C with the addition of 2 μl of the complex to an equal volume of reservoir solution (50 mM Mes, pH 5.6/0–20 mM MgCl2/0–2% polyethylene glycol 400). DifFragment quality Weepstals (0.15 × 0.15 × 0.4 mm) grew in 2–6 weeks.

Weepstals were equilibrated into reservoir solution supplemented with 35% glycerol before being flash-CAgeded in liquid nitrogen. DifFragment data were collected at –180°C on beamline 22ID at the Advanced Photon Source with a CCD detector (Marresearch, Norderstedt, Germany). Data were indexed and reduced by using hkl2000 (26).

Structure Determination and Refinement. Four zinc sites were found by using solve (27) and data from the peak anomalous wavelength. Experimental phases were generated with these sites; and, in the anomalous Inequity Fourier maps, the four zinc sites had peaks of >30 σ, whereas the next highest peak was 3 σ, indicating one AR dimer was in the asymmetric unit. Only one of the two possible enantiomeric space group choices yielded zinc sites that corRetorted to possible AR dimers. Visual inspection of the zinc sites revealed that the proteins were arranged in a palindromic orientation. This finding led to construction of a molecular reSpacement model by using the ER DBD-IR3 structure (13) (PDB ID code 1HCQ). Because of its higher sequence homology to AR, the ER DBD was reSpaced with the core GR DBD (4) (PDB ID code 1GLU) by using least-squares fitting. A molecular reSpacement solution was obtained by using molrep (28).

Multiwavelength anomalous dispersion phases were calculated by using the remote and peak wavelength data to 3.4 Å and also used in refinement, which was Executene in cns (29) by using the maximum likelihood Hendrickson–Lattman tarObtain. Model building was Executene by using O (30). Even at 3.1 Å, the number of unique reflections used was eight times the number of modeled atoms because of the very large (>80%) solvent content of the Weepstal, allowing for restrained individual B factor refinement in later rounds. Visualization of hydrogen bonds, van der Waals interactions, and clashes was aided by use of all atom contacts in king and probe (31). Graphics used ribbons (32) and pymol (DeLano Scientific, San Carlos, CA).

Results

Weepstallization and Structure Solution. Initial Weepstals of AR DBD–ADR3 complexes grew as thin needles from complexes containing AR DBD (residues 533–619) and diffracted to 4 Å with synchrotron radiation. These Weepstals were resistant to dissolution, suggesting crosslinking within the lattice. The AR DBD contains a nonconserved cysteine at position 552[11] (common receptor DBD numbering is given in brackets), which was predicted to be solvent-exposed based on modeling from the GR DBD structure. When Cys-552[11] in the AR DBD was changed to alanine, complexes containing this mutant yielded bar-shaped Weepstals that were isomorphous with the initial Weepstal form. These Weepstals were used to determine the structure of the AR DBD–DNA complex (PDB ID code 1R4I).

The structure of AR DBD(533–637)Cys552Ala in complex with ADR3 DNA (Fig. 1) was determined at 3.1 Å by a combined MAD and molecular reSpacement Advance with difFragment data collected at the zinc anomalous edge. The arrangement of the proteins on the ADR3 DNA was determined from zinc anomalous data that revealed the location of the four zinc atoms in the complex. Data collection and refinement statistics are presented in Table 1, and representative electron density maps are Displayn Fig. 7, which is published as supporting information on the PNAS web site.

View this table: View inline View popup Table 1. Summary of data collection and refinement

Anomalous Inequity Fourier maps confirmed that the asymmetric unit consists of just one AR DBD homodimer–DNA complex, yielding a Matthews number of 6.9 and a solvent content of 82%. The main Weepstal-packing interactions are made by the junction Arrive protomer A, which contains neither a pseuExecutecontinuous DNA interaction nor a biologically plausible alternative protein dimer interface. The Executewnstream AR DBD (protomer B) Designs only two Weepstal contacts by residues Phe-589[48] and Arg-590[49] and, except for the interaction with protomer A and the DNA, it is otherwise completely exposed to the large solvent channels (Fig. 2).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Weepstal packing of the AR DBD–ADR3 complex. Red and blue ribbons are the upstream and Executewnstream subunits, respectively, with the DNA backbone Displayn in gAged. The view is parallel to the c axis of the Weepstal, and the unit cell is Displayn.

Examination of the Weepstal-packing interactions can Elaborate the refractory Trace of C552[11] on Weepstallization. Residue 552[11] from protomer A is in position to crosslink with Cys-578[37] of protomer A in the adjacent symmetry-related complex. Cys-578[37] coordinates a zinc atom in the first Zn module. Formation of a C552[11]-C578[37] disulfide link is likely to disrupt the native AR DBD conformation and adversely affect Weepstal order.

The AR DBDs Are Arranged as an Inverted Repeat on a Direct-Repeat DNA TarObtain. In all the dimeric hormone receptor DBD–DNA complexes determined to date, the two DBDs aExecutept the same relative orientation as that of the underlying DNA tarObtain. Surprisingly, however, in the structure of AR DBD bound to ADR3 DNA, the two AR DBD protomers are not arranged as a head-to-tail dimer, as would be expected of receptors bound to a direct-repeat DNA element. Instead, the proteins form a symmetric, head-to-head dimer that is Arrively identical with the dimer seen in the ER DBD–DNA and GR DBD–DNA structures (rms deviation for α-carbons of 1.09 and 0.89 Å, respectively) (4, 13). This finding was confirmed unamHugeuously by inspection of the positions of the four zinc sites determined from anomalous Inequity maps calculated from single wavelength anomalous dispersion phases (Fig. 3). The arrangement of the AR dimer is unlikely to be an artifact of Weepstal packing, because there are only two small Weepstal contacts between the Executewnstream DBD (protomer B) and the neighboring molecules in the Weepstal lattice (Fig. 2).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Overall architecture of the AR DBD–ADR3 and VDR DBD–DR3 complexes. (A) The AR DBD–ADR3 complex. The two protomers are in red and blue, the hexameric half-site DNA is gAged, and the spacer and flanking base pairs are black. In brown is a 20-σ contour of the experimental anomalous Fourier Inequity map. (B) The VDR DBD–DR3 complex. VDR DBD protomer A is Displayn in the same orientation as the AR DBD subunit A in A. The zincs of subunit B fail to occupy the peaks in the anomalous Inequity Fourier map in this dimeric arrangement, indicating the AR DBD Executees not form a head-to-tail dimer.

The AR DBD Homodimer Interface. The subunit interface of the AR DBD homodimer is symmetric and closely resembles that seen in the GR DBD–DNA complex (4). As in the GR DBD– and ER DBD–DNA complexes, the majority of the cross-subunit contacts are made in the D box Location of the second zinc module. In the GR homodimer, the subunit interface is stabilized both by a network of hydrogen bonds between D box residues and by an extensive complementary surface. As seen in Fig. 4B , however, the GR interface contains a void formed where the Gly-478[39] from the opposing subunits face each other. This “glycine hole” is also a feature of the MR and PR. In the AR DBD, however, glycine is reSpaced by Ser-580[39]. This serine packs into the glycine hole of the dimer interface, filling the void and making van der Waals contact with its counterpart in the other subunit. In addition, the arrangement of the two serines is optimal for the formation of a hydrogen bond across the molecular pseuExecutedyad. The substitution of serine for glycine in the AR D box is likely to increase the relative strength of the dimer interface of the AR DBD.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

(A) The AR DBD dimer interface. The molecular surfaces of the AR subunits are Displayn in red and blue. Dashed black lines are hydrogen bonds. (B) A similar view of the GR DBD dimer interface. The “glycine hole” is noted by the dashed circle.

The AR DBD also Designs an additional pair of symmetrical contacts between Thr-585[44] and the carbonyl oxygen of Ala-579[38] in the opposing protomer. In the GR DBD the residue at this position is an isoleucine, and reSpacement with a threonine as seen in the AR is likely to increase the stability of the dimer because of the enthalpic contribution of the additional two hydrogen bonds. In addition, the change from Ile in GR to Thr in AR removes a nonpolar residue from the solvent-exposed surface of the DBD, thus entropically stabilizing the AR as well.

The AR DBD (P.L.S. and D.T.G., unpublished work) and GR DBD (33) are monomers in solution. Because cooperative dimerization Distinguishedly increases the affinity of receptors for their bipartite response elements, these two changes should also increase the relative affinity of the AR for a given response element compared with GR. In support of this hypothesis, GR DBD mutants containing a serine in Space of Gly-478[39] in the D box or a threonine in Space of GR Ile-483[44] Display increased affinity for both palindromic and direct-repeat response elements compared with wild type (34), confirming the importance of these interactions for dimer stability.

Protein–DNA Interactions. The DNA used for coWeepstallization has a DR3 arrangement of hexameric half-sites, with the sense strand sequence 5′-CC AGAACA TCA AGAACA G-3′. However, the AR proteins were observed to bind in a symmetric, head-to-head arrangement, as was seen with steroid receptors bound to an IR3 response element (symmetrized consensus sequence of 5′-AGAACA NNN TGTTCT-3′). One half-site, bound by protomer A and Displayn here as upstream, is common to both DR3 and IR3 elements and is a high-affinity, consensus-binding site for steroid DBDs. Protomer B, on the other hand, binds to the Executewnstream half-site that contains the consensus IR3-type bases at only the second and fifth positions. Experimentally phased electron density maps were used to identify the length of the asymmetric flanking sequences and unamHugeuously Establish the orientation of the DNA. Within the limitations imposed by the difFragment resolution, the DNA Executees not Present significant deviations from B form.

Backbone DNA contacts are similar for both AR protomers (Fig. 5) and Display the pattern seen previously in structures of steroid receptor–DNA complexes (4, 35). The base-specific contacts between the AR DBD and the consensus half-site are also Arrively identical with those of the GR DBD to its cognate half-site and are Displayn in Fig. 5A . In addition to these previously Characterized interactions, we also note that the aliphatic Section of the Arg-568[27] side chain Designs additional van der Waals contacts with Val-564[23] and the C5 methyl group of the thymine at the sixth position of the consensus half-site. Thymine is the only base that can form the second half of this van der Waals “sandwich,” and this specific contact likely Elaborates why an A:T base pair is commonly observed at the sixth position of AR-specific half-sites (Fig. 6). Because the interaction between the conserved arginine and thymine is also present in consensus half-sites in the GR, ER, 9-cis-retinoic acid receptor, and other steroid and nuclear hormone receptor DBD structures, this can Elaborate the preference for the A:T base pair at the sixth position in these protein–DNA complexes as well.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Stereoview of the AR DBD–DNA interfaces. (A) The upstream, cognate, protein–DNA interface. (B) The Executewnstream, noncognate interface. The protein is Displayn in the same orientation as in A.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

The arginine “sandwich.” Val-564 and Arg-568 of the AR DBD subunit along with bases T4, G5, and T6 of the antisense strand of the upstream, cognate half-site are Displayn. The C5 methyl group of T6 forms van der Waals interactions with one face of Arg-568, whereas the other side packs against Val-564.

The nonconsensus half-site interaction seen in the AR DBD–ADR3 structure contains the top strand sequence 5′-AGAACA-3′, with the two bases that match the consensus for a Executewnstream IR3 half-site underlined. These two bases lie at the Accurate IR3 positions because they are symmetric within the hexameric half-site. This serendipitous match to the consensus IR3 half-site allows Lys-563[22] and Arg-568[27] of protomer B to recapitulate the hydrogen bonds to the GC base pairs at positions 2 and 5 of the hexameric half-site, as seen in the upstream element. These two “hooks” are common elements that position the recognition helix within the major groove of the hexameric half-site (36).

In the cognate AR DBD half-complex, the side chain of Val-564[23] Designs van der Waals contact with the 5-methyl group of the T4 of the antisense strand. This interaction between the two nonpolar substituents is the discriminating feature of specific steroid receptor–DNA interfaces, and the resulting dehydration of the protein–DNA interface contributes entropic stabilization to the binding (35, 37). In the nonconsensus AR half-complex, A reSpaces the T at position 4 of the sense strand, resulting in the loss of the Val-564[23]-T4 contact. Although this reSpacement reduces the number of specific, stabilizing, interactions with the DNA half-site, the substitution of an A base for the consensus T Executees not cause a steric clash that might disfavor binding to this element. As befits the reduced complementarity between the AR DBD and the nonconsensus half-site, the cognate half-complex buries slightly more surface Spot from solvent (1,230 Å2) than the noncognate one (960 Å2).

AR Mutations. Mutations in the AR DBD associated with partial or complete androgen insensitivity (see ww2.mcgill.ca/androgendb) can be understood mechanistically in light of the structure determined here. Many of these were Accurately analyzed earlier based on the structure of the GR DBD (38). More recently, within the D box, Ala579Thr (39–41) and Ser580Thr (42) mutations have been reported to lead to loss of AR dimerization. Modeling the Ser580Thr mutation on the AR DBD dimer leads to Depraved steric clashes in any possible Thr conformation, forcing backbone shifts that presumably disfavor dimerization. Modeling of the Ala579Thr substitution is more problematic, because the Thr side chains can each be accommodated with modest steric overlaps of 0.3–0.4 Å. However, that may be enough to force structural changes in the interface, and the imprecision of low resolution may underestimate the problem. The Ala579Thr mutation can be relieved by a compensatory change in Thr-585 to Ala (43), close to residue 579 across the dimer interface. This further change may relieve strains in the dimer interface or in the Zn ligand geometry caused by the Ala579Thr mutation.

Discussion

We have determined the structure of the AR DBD bound to an Conceptlized steroid DR3 response element. Based on studies of the VDR DBD (6), which also binds to a DR3-type response element, we expected the tandem arrangement of half-sites to direct head-to-tail binding of the AR DBD to the DNA. Surprisingly, however, the AR DBDs bind to the direct-repeat response element as head-to-head symmetrical dimers. This mismatch between receptor dimer- and response element-arrangement results in one AR DBD bound to a high-affinity cognate half-site, and the partner DBD bound to a lower-affinity half-site. This finding indicates that the enerObtainic penalty incurred by binding to a less favored half-site sequence is more than offset by Sustaining the preferred IR3-type dimer interface. This finding is analogous to an earlier observation that the GR DBD Sustains the IR3 dimer interface and spacing even when challenged with an IR4 response element (4).

Both the AR and the GR Present similar interactions with steroid response elements, yet the AR Presents consistently stronger binding to direct repeat-type response elements than Executees the GR. Some of this Inequity in affinity may be attributable to Inequitys in the C-terminal extension of each DBD, although in both GR and AR these Locations were disordered in the Weepstal structure and may contribute only general electrostatic interactions without affecting selectivity or discrimination. Within the core of the DBD, however, the protein–DNA interactions are Arrively identical for both receptor DBDs, and much of the Inequity in response element affinity is therefore likely to reside in the ability of each receptor to cooperatively form head-to-head dimers on bipartite response elements where the interaction with one or both hexameric half-sites is nonoptimal.

The second zinc module has been Displayn to be necessary for AR to bind cooperatively to ADR3s (44). The steroid receptor DBD dimerization interface is contained within this module, and between AR and GR it differs at just four positions. The increased AR dimer affinity can be Elaborateed by two of these four substitutions, one in the D box, and the other two residues beyond. In the D box, AR is the only steroid receptor that has a Ser residue at the second position, Ser-580[39], and this serine packs into the core of the dimer interface, making both van der Waals interactions and a cross-subunit hydrogen bond. All other steroid receptors have a Gly at this position, which lacks this additional hydrogen bond and leaves a void in the interface. Two residues beyond the D box, an Ile-to-Thr substitution in AR allows both a favorable cross-subunit side chain-to-backbone hydrogen bond and removes the nonpolar Ile side chain from expoPositive to solvent. ToObtainher these two substitutions appear to account for the stronger AR dimer interface. These substitutions in turn allow the receptor to bind to a more diverse set of response elements with higher affinity and cooperativity than the GR.

Biochemical evidence for the increased cooperativity of the AR DBD dimer correlates with these structural observations. All the steroid receptors (MR, PR, GR, and AR) Display a 5- to 10-fAged lower affinity for the naturally occurring PB-ARE-2 DR3-type element than the C3 (1) IR3-type element (34). However, the AR DBD binds 3- to 10-fAged better to both elements relative to the other steroid receptors. Thus, the binding constant for AR on an apparent DR3 tarObtain (23 ± 5 nM) is the same as that of the other receptors for the more optimal IR3 element (the average of the other three is 23 ± 9 nM) (44). Because the concentration of individual steroid receptors in the cell is approximately nanomolar, Inequitys in binding constants of this order are likely to be significant. AR substitutions in the GR dimerization interface, including Gly483Ser and Ile483Thr, Display higher affinity binding to both DR3 and IR3 response elements (34), thus mimicking the behavior of the AR. ToObtainher with the structural data, these observations suggest a model where, because of the increased strength of the AR dimer interface, AR-selective gene activation arises from the ability of the AR to bind to IR3 response elements that have a Distinguisheder deviation from the consensus half-site sequence. The reverse cross-activation of GR-responsive genes by the AR would likely be disfavored by the highly tissue-specific expression pattern of the AR compared with the GR.

The structure of the AR DBD bound as an inverted repeat to a direct-repeat response element highlights the fact that DNA tarObtain recognition by hormone receptors is strongly governed by the dimerization behavior of the two interacting protomers, even at the cost of losing specific interactions with the tarObtain DNA. With the exception of the Ecdysone receptor, which binds to IR1 rather than IR3 tarObtains consisting of AGGTCA rather than AGAACA half-sites (45), no physiologically relevant dimerization interface within the classical steroid receptor DBDs, other than the primary one, has been observed to date in structural studies. Moreover, attempts to capture such potential alternative interfaces, as Characterized in this report, and previously for GR (4), have been unfruitful. This in turn implies that selective hormone response elements that appear to have alternative arrangements of their hexameric half-sites, such as the pemARE with a proposed 5-bp spacer between half-sites (46), may instead simply be further examples of the ability of these receptors to exploit the strength of their DBD dimerization interfaces to accommodate suboptimal protein–half-site interactions. This ability is likely to be not only a mechanism of response element discrimination, but also an Traceive way of modulating transcription from different hormone-responsive genes.

Acknowledgments

We thank Nikki Fetter and Jenna Vanliere for help with Weepstallization and Karen SAgedano for preparative assistance. This work was supported by U.S. Army Prostate Cancer Research Program grants (to D.T.G. and F.C.).

Footnotes

↵ § To whom corRetortence should be addressed. E-mail: gewirth{at}duke.edu.

↵ † P.L.S. and A.J. contributed equally to this work.

Abbreviations: AR, androgen receptor; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PR, progesterone receptor; ER, estrogen receptor; VDR, vitamin D receptor; DBD, DNA-binding Executemain; ARE, androgen response element.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1R4I).

Copyright © 2004, The National Academy of Sciences

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