A Progesterone Receptor Co-activator (JDP2) MediatesActivity through Interaction with Residues in theCarboxyl-terminal Extension of the DNA Binding Domain□S �

Received for publication, April 6, 2009, and in revised form, June 19, 2009 Published, JBC Papers in Press, June 24, 2009, DOI 10.1074/jbc.M109.003244

Krista K. Hill‡, Sarah C. Roemer§, David N. M. Jones‡§, Mair E. A. Churchill‡§1, and Dean P. Edwards¶

From the ‡Molecular Biology Program and §Department of Pharmacology, School of Medicine, University of Colorado Denver,Aurora, Colorado 80045 and the ¶Departments of Molecular and Cellular Biology and Pathology, Baylor College of Medicine,Houston, Texas 77030

Progesterone receptor (PR) belongs to the nuclear receptorfamily of ligand-dependent transcription factors and mediatesthemajor biological effects of progesterone. Transcriptional co-activators that are recruited by PR through the carboxyl-termi-nal ligand binding domain have been studied extensively. Muchless is known about co-activators that interact with other re-gions of receptors. Jun dimerization protein 2 (JDP2) is a PRco-activator that enhances the transcriptional activity of theamino-terminal domain by increasing the�-helical content andstability of the intrinsically disordered amino-terminal domain.To gain insights into the mechanism of JDP2 co-activation ofPR, the structural basis of JDP2-PR interaction was analyzedusing NMR. The smallest regions of each protein needed forefficient protein interaction were used for NMR and includedthe basic region plus leucine zipper (bZIP) domain of JDP2 andthe core zinc modules of the PR DNA binding domain plus theintrinsically disordered carboxyl-terminal extension (CTE) ofthe DNA binding domain. Chemical shift changes in PR upontitration with JDP2 revealed that most of the residues involvedin binding of JDP2 residewithin theCTE. The importance of theCTE for binding JDP2 was confirmed by peptide competitionand mutational analyses. Point mutations within CTE sitesidentified byNMRand aCTEdomain swapping experiment alsoconfirmed the functional importance of JDP2 interaction withthe CTE for enhancement of PR transcriptional activity. Thesestudies provide insights into the role and functional importanceof the CTE for co-activator interactions.

Progesterone receptor (PR)2 is a member of the nuclearreceptor (NR) family of ligand-activated transcription factors

that regulate a variety of biological processes by binding to spe-cific progesterone-response elements (PREs) and activating orrepressing expression of target genes (1–3). Nuclear receptorsare modular proteins consisting of a highly conserved DNAbinding domain (DBD), a less well conserved carboxyl-terminalligand binding domain (LBD), and a poorly conserved amino-terminal domain (NTD) that is required formaximal transcrip-tional activity. NRs have at least two transcription activationfunctions, constitutively active AF1 in the NTD and ligand-de-pendent AF2 in LBD (4). The LBDs andDBDs of nuclear recep-tors are well ordered, and high resolution structures have beensolved; however, no structures of the NTD have been deter-mined (5–8). Structures of the NTD have been a difficult chal-lenge because it is largely an intrinsically disordered protein(IDP) domain (9, 10). IDPs consist of amino acids with lowsequence complexity and a high proportion of charged residueswith few hydrophobic residues, resulting in long stretches ofrandom coil and only a few short segments of �-helix. IDPs donot spontaneously fold into classical globular domains but canundergo a disorder-order transition upon binding target pro-teins or DNA (11–15). Coupled folding and binding are advan-tageous because they enable a single regulatory protein to inter-act with a wide variety of binding partners, and the low affinityof IDPs is ideal for transient protein-protein and protein-DNAinteractions (13).A short non-conserved 40–50-amino acid segment between

the DBD and the LBD, termed the carboxyl-terminal extension(CTE), also has hallmarks of an IDP including a high density ofbasic residues, little secondary structure, and random coil.Work from our group and others has shown that the CTE canparticipate in DNA binding (16–26). A crystal structure of thePR DBD-CTE�DNA complex revealed that the CTE forms anextended loop that interacts with the minor groove flankingeither side of the PRE. Mutational analysis confirmed theimportance of minor groove-interacting residues in the CTEfor high affinity binding to PREDNA (21). Thus, theDNAbind-ing domain of PR is bipartite, consisting of core zinc fingermodules that bind specific hormone-response elements in themajor groove and the CTE that binds less specifically to theflanking minor groove (see Fig. 1A). The CTE of steroid recep-tors also binds the chromatin high mobility group proteins-1and 2 (HMGB-1/-2). Interactions with HMGB-1/-2 increasethe affinity of steroid receptors for their target DNA through a

� This article was selected as a Paper of the Week.□S The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. 1 and 2 and supplemental Table 1.1 To whom correspondence should be addressed: University of Colorado

Denver, 12801 E. 17th Ave., P. O. Box 6511, MS 8303, Aurora, CO 80045. Tel.:303-724-3670; E-mail: mair.churchill@ucdenver.edu.

2 The abbreviations used are: PR, progesterone receptor; PRE, progesterone-response element; NR, nuclear receptor; TR, thyroid receptor; ER, estrogenreceptor; PPAR, peroxisome proliferator-activated receptor; RXR, retinoidX receptor; LBD, ligand binding domain; DBD, DNA binding domain; NTD,amino-terminal domain; CTE, carboxyl-terminal extension; sCTE, scram-bled CTE; IDP, intrinsically disordered protein; HMGB, high mobility groupprotein; CREB, cAMP-response element-binding protein; bZIP, basic regionplus leucine zipper; aa, amino acids; GST, glutathione S-transferase; DTT,dithiothreitol; HSQC, heteronuclear single quantum correlation; TOCSY,total correlation spectroscopy.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 36, pp. 24415–24424, September 4, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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transient protein-protein interaction that relieves a repressiveeffect of the CTE on DNA binding (27–35).The AF2 region of nuclear receptors interacts with the p160

family of steroid receptor co-activators through an LXXLLmotif that recognizes a specific complementary hydrophobiccleft in the well structured LBD. The structural basis for ligand-dependent interactions of p160 co-activators with AF2 hasbeenwell characterized by high resolution co-crystal structuresof various LBD�LXXLL peptide complexes (5, 36–38). Muchless is known about proteins that interact with and mediatetranscriptional activity of the NTD. Consistent with an intrin-sically disordered region, awide range of co-regulatory proteinshas been reported to interact with the NTD (9, 10, 39). Studieshave also demonstrated that the NTD of various steroid recep-tors interacts with components of the general transcriptionmachinery (TATA binding protein, transcription factor IIH) orwith the co-activator CREB-binding protein and that theseinteractions induce a more ordered folded state of the NTD(40–45). These results have led to the idea that protein-in-duced folding of the NTD mediates AF1 activity.We previously identified Jun dimerization protein 2

(JDP2) as a PR-interacting protein that enhances the tran-scriptional activity of the NTD and can act in a manner inde-pendent of the LBD AF2 (46–48). JDP2 is a basic DNA bind-ing domain plus leucine zipper (bZIP) protein that lacks anamino-terminal activation domain (49). Interaction ofendogenous JDP2 and PR in T47D breast cancer cells wasdetected by co-immunoprecipitation assay, and the chroma-tin immunoprecipitation assay demonstrated a hormone-dependent co-recruitment of JDP2 and PR to the proximalPRE-containing promoter of anMMTV reporter gene stablyintegrated in T47D cells (47). Mapping studies identified theDBD plus CTE as the minimal JDP2 binding region withinPR (46, 47). Results from circular dichroism, partial proteol-ysis, and functional mutagenesis experiments demonstratedthat JDP2 interaction promotes a more ordered structure ofthe NTD in a manner that correlates with enhanced tran-scriptional activity of the NTD (48). Because JDP2 interac-tion occurs with the DBD and not directly with the NTD, thissuggests that its effect on PR transcriptional activity is prop-agated through an interdomain communication between theDBD and the NTD.The goal of this workwas to further define themechanism by

which JDP2 interacts with PR to modulate transcriptionalactivity of theNTD. Several approacheswere used tomap func-tionally important JDP2 interaction sites in PR, includingNMRchemical shift perturbations, and the effects of mutations onJDP2-PR protein interactions and PR transcriptional activity.Specific residues in a subregion of the CTE were defined asmost critical for JDP2 binding and for co-activation of PR,whereas other residues in the CTE and core DBD appeared toundergo a conformational change or a change in their chemicalenvironment. These data implicate a role for the CTE as a bind-ing site for co-activator proteins and provide insights into themechanism of interaction of co-activators that bind to regionsof steroid receptors other than AF2 in the LBD.

EXPERIMENTAL PROCEDURES

Plasmids—PR expression vectors and reporter plasmidshave been described previously including phPR-B expressingfull-length human PR-B under the control of the SV40 pro-moter. pRL-SV40 (Promega, Madison,WI) containing a cDNA(Rluc) encoding Renilla luciferase was used as an internal con-trol reporter. A reporter gene construct PRE2-TATA-LUCwas described previously (48). Amino-terminal glutathioneS-transferase (GST)-tagged JDP2 constructs were constructedby PCR amplification of full-length cDNAs with primers con-taining unique 5� and 3� restriction sites for insertion intoBamHI and EcoRI sites of pGEX-2T. JDP2 constructs included:full-length rat JDP2 (aa 1–163), JDP2 amino-terminal plus basicregion (aa 1–96), JDP2 bZIP region (aa 69–137), JDP2 basicregion (aa 74–96), and JDP2 leucine zipper region (ZIP) (aa105–137). GST-tagged full-length rat JDP2 (aa 1–163; 18.8kDa) was created from a pCDNA his-rJDP2 vector from A.Aronheim (Technion-Israel Institute of Technology). pCR3.1rat JDP2, under the control of the cytomegalovirus promoter,was constructed by insertion of JDP2 cDNA into BamHI andEcoRI sites of the pCR3.1 vector (Invitrogen).Hormones, Antibodies, andWesternBlots—Progesteronewas

obtained from Sigma. Antibodies included a rabbit polyclonalprepared against the full-length rat JDP2 protein and a PRDBDpolyclonal antibody as described previously (31). Proteinswere separated on 12 or 15% SDS-polyacrylamide gel elec-trophoresis and analyzed by Western blotting as describedpreviously (29, 31, 48). Detection was enhanced by chemilu-minescence (Millipore).Bacterial Expression of Recombinant JDP2 and PR DBD—

JDP2 was overexpressed in Escherichia coli strain BL21 andcells were grown at 37° to anA600 of 0.8 after protein expressionwas induced by 1 mM isopropyl �-D-thiogalactopyranoside for4 h. Cells were harvested, and the pellet was resuspended (20mM Tris-HCl, pH 7.4, 25% sucrose, 2 mMMgCl2) and lysed in adetergent buffer (4mMEDTA, 0.2 MNaCl, 1% deoxycholic acid,1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5) containinglysozyme and DNase I. The clarified lysate was incubated withglutathione-Sepharose 4B (Amersham Biosciences) for 3 h at4 °C. After extensive washing of the resin with a high salt wash(3 M NaCl) and equilibration in thrombin cleavage buffer (20mMTris, pH 8.0, 250mMNaCl, 10% glycerol, 1 mMDTT), JDP2was cleaved from the resin with thrombin at 4 °C for 12–16 h,concentrated, and further purified using a cation exchangeSource 15S column (GE Healthcare). GST-tagged PR DBD wasoverproduced in E. coli strain BL21 (DE3) using the pGEX-2T overexpression system. Cells were grown at 37 °C in Luriabroth. When the A600 reached 0.8, protein expression wasinduced by the addition of 1 mM isopropyl �-D-thiogalactopy-ranoside, and cells were harvested after 4 h. The cells were lysedin a buffer containing 1� BugBuster (Novagen), 5 mM DTT,and Benzonase and protease inhibitors, and the cell extract wascleared by centrifugation. The cell lysate was then incubatedwith glutathione-Sepharose beads for 3 h at 4 °C, and the beadswere washed repeatedly in a buffer containing 20 mM Tris, pH7.5, 1 M NaCl, 100 �M ZnCl, 1 mM EDTA, 10% glycerol, and 1mM DTT and then cleaved overnight in a buffer containing 20

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mM Tris, pH 7.5, 100 mM NaCl, 2.5 mM CaCl2, 10% glycerol, 1mM DTT, and 400 units of thrombin. Cleaved protein wasloaded onto a Resource S cation exchange column (GE Health-care, Upchurch) in a buffer containing 20 mM Tris, pH 7.5, 100mM NaCl, 1 �M ZnCl, 10% glycerol, 1 mM EDTA, and 1 mM

DTT, eluted with a linear salt gradient to 750 mM NaCl, andthen dialyzed into the same buffer with 100 mM NaCl.Cell Culture and Transfection—Cos-1 cells were maintained

in Dulbecco’s modified Eagle’s medium supplemented with 5%fetal bovine serum (HyClone-Millipore, Billerica, MA). Cellswere plated in 6-well dishes at a density of 1.6 � 105 cells/well.At 24 h after plating, Cos-1 cells were transfected using Lipo-fectamine Plus reagents (Invitrogen) according to themanufac-turer’s instructions. Following transfection, cells were grown inDulbecco’s modified Eagle’s medium supplemented with 5%charcoal-stripped fetal bovine serum for 24 h and then treatedin the same medium with 10 nM progesterone.In Vitro GST Pulldown Protein Interaction Assays—Bacterial

cell lysates containing free GST protein or GST�JDP2 fusionproteins, as described previously (47), were incubated with 50�l of a 50% slurry of glutathione-Sepharose resins (GE Health-care) in phosphate-buffered saline for 1 h at 4 °C. Resins werethen washed three times (10 mM Tris-HCl, pH 7.8, 100 mM

NaCl, 10% glycerol, 2 mMMgCl2, 1 mM EDTA, 1 mMDTT, and100�g/ml bovine serum albumin), brought to a total volume of250 �l in wash buffer, and incubated with PR DBD protein for1 h at 4 °C. Resins were then washed extensively, and the boundproteins were eluted with 2% SDS-�-mercaptoethanol samplebuffer and detected by Western immunoblotting. In peptidecompetition assays, 1 �g of purified PR DBD-CTE650 was incu-bated with CTE peptide or scrambled CTE (sCTE).Cell-based Receptor Transactivation Assays—Cos-1 cells

were plated at 1.6 � 105 cells/well in six-well culture dishes. At24 h, cells were transfectedwith constitutively active pRL-SV40(Promega) Renilla luciferase expression vector as an internalcontrol for transfection efficiency, PRE2-TATA-Luc progester-one-responsive luciferase reporter plasmid, PR or PR mutantexpression vectors, and varying amounts of pCR3.1-JDP2 vec-tor, as described previously (48). pCR3.1 empty vector wasadded to maintain a constant amount of cytomegalovirus pro-moter. At 48 h after transfection, cells were washed once inphosphate-buffered saline and then lysed using the Dual-Lucif-erase reporter assay system kit (Promega) according to themanufacturer’s instructions. Luciferase assays were performedaccording to the manufacturer’s protocol using 15 �l of celllysates. Firefly luciferase activity was normalized to Renillaluciferase activity, and relative firefly luciferase activity was cal-culated by setting the normalized values obtained with vehicle-treated reporter alone (nohormone, noPR) to 1.0 and setting allother values as a -fold increase relative to 1.0. The -fold increasefirefly luciferase induction was calculated as a ratio of relativefirefly luciferase activity of hormone-treated samples dividedby relative luciferase activity of corresponding vehicle-treatedsamples. At least three independent experiments were per-formed, and values for luciferase expression were calculated asaverages � S.E.NMR Studies—GST-tagged 13C15N-labeled PR DBD-CTE

(aa 562–641) was expressed in E. coli strain BL21 (DE3) using

the pGEX-2T expression system (Novagen). Cells were grownat 37 °C in aminimalmedium containing 1 g of 15NH4Cl and 2 gof 13C-glucose in 1� M9 salts supplemented with 2 mM

Mg2SO4 and 0.1 mM CaCl2/liter medium. When the A600reached 0.5, protein expression was induced by the addition of0.5 mM isopropyl �-D-thiogalactopyranoside, and cells wereharvested after 4 h.Theproteinwas purified as described above.The collected fractions were pooled and dialyzed into a buffercontaining 20 mM Tris, pH 7.5, 100 mM NaCl, 1 �M ZnCl, 5%glycerol, 1 mM EDTA, and 1 mM DTT and concentrated to 400�M. The NMR sample was combined with 10% D2O and twogranules of 2,2-dimethyl-2-silapentane-5-sulfonic acid as a ref-erence. NMR spectra were recorded at 15 °C on a Varian 600-MHz spectrometer using a (5-mm) triple-resonance (1H/13C/15N) pulsed field gradient cold probe. The following spectrawere recorded: HSQC, HNCACB, TOCSY-CCONH, TOCSY-HSQC, CACBCONH, and HAHBCONH. NMR data were pro-cessed using NMRPipe (50) and analyzed using SPARKY (51).1H chemical shifts were referenced to 2,2-dimethyl-2-silapen-tane-5-sulfonic acid, and 15N and 13C chemical shifts were ref-erenced indirectly from the gyromagnetic ratios.

RESULTS

JDP2 Interaction Perturbs Residues in the Core DBDandCTEas Detected by NMR—Our previous studies mapped the regionof PR that binds JDP2 to the PR DBD plus CTE and the bZIP astheminimal portion of JDP2 (47). To further narrow the regionof JDP2 that interacts with PR, various GST�JDP2 constructs(Fig. 1B) were used in pulldown assays including full-lengthJDP2 (aa 1–163), the amino terminus plus basic region (aa1–96), basic leucine zipper region (aa 69–137), basic regionalone (aa 74–96), and leucine zipper region alone (aa 105–137).GST�JDP2 constructs or free GST were immobilized to gluta-thione-Sepharose resins and incubated with a PR DBD con-struct that also contains amino acids 632–641 of the CTE (PRDBD-CTE641). PR DBD-CTE641 bound to all of the GST�JDP2constructs with the exception of the leucine zipper (Fig. 1C).Thus, the smallest region of JDP2 that interacted with the PRDBD-CTE641 was the basic domain. Although smaller domainsor fragments are theoreticallymore amenable forNMR studies,the basic region alone was less stable during purification thanother fragments. Therefore, the bZIP was expressed and puri-fied for NMR studies.Because JDP2bZIP andPRDBD-CTE641 are small and can be

expressed in bacteria and purified as soluble proteins at highconcentrations (5mg/ml), the PR�JDP2 complex is amenable toNMR analysis. 13C 15N-labeled PR DBD-CTE641 was producedfor NMR experiments as described under “Experimental Pro-cedures.” This construct is similar to that used previously forcrystallography, which was sufficient for visualization of theCTE interactionwith theminor groove of DNA (21). A series ofNMR spectra were recorded including HSQC, HNCACB,TOCSY-CCONH, TOCSY-HSQC, CACBCONH, and HAHB-CONH. Backbone chemical shift assignments were made, andthe labeled resonance assignments of the amide nitrogen andhydrogen atoms for each amino acid are shown in the 15N-HSQC spectrum (Fig. 2A and supplemental Table 1). Compar-ison of the chemical shift values with the predicted chemical

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shifts from the crystal structure of the PR DBD�DNA complex(21) demonstrates that there are no major conformational dif-ferences between the free and DNA-bound protein with theexception of a few residues in the �-hairpin, the end of helix 1,and the CTE (52–56) (supplemental Fig. 1). This implies thatthe DBD-CTE in solution takes on a slightly different confor-mation than the DBD-CTE bound to DNA. To investigate thisfurther, the chemical shift assignments for PR DBD-CTE641

were submitted to an online structure prediction server,THRIFTY (56, 57). The predicted three-dimensional structureof DBD-CTE641 in solution is similar to the crystal structure ofPRDBD-CTE641 bound toDNA (21), except for an altered posi-tion of the CTE. These data implicate the CTE as a flexibleregion that is capable of adopting a different conformationwhen bound to a partner macromolecule (supplemental Fig. 2).Chemical shift perturbations were used to identify potential

sites of PR that interact with JDP2.As increasing amounts of unlabeledJDP2 bZIP were titrated into 15N-labeled PR DBD-CTE641 and suc-cessive HSQC spectra were re-corded, chemical shift changes inseveral resonances were noted. Theresidues that shift or disappear withthe addition of JDP2 bZIP areshown in the overlay and enlarge-ment of the HSQC spectra in Fig.2B. For example, resonances corre-sponding to Gly635 andGly636 in theCTE disappeared upon the additionof JDP2, whereas residues Arg637and Lys638 underwent a chemicalshift change (Fig. 2B). Chemicalshift changes were also observed inthe core DBD second zinc finger,most notably Gly585 at the begin-ning of the DNA recognition helixand Arg593 at the end of the helix, ahydrophobic patch around Met595andGly597, andAsp612 and Lys613 inthe dimerization box (Fig. 2B).Using the chemical shift assign-

ments made with the double-la-

FIGURE 1. The basic domain of JDP2 is the minimal region to interact with PR. A, schematic of the nuclearreceptor DBD showing the Gly-Met (GM) boundary distinguishing the core DBD from the CTE. Open circles representamino acids, and light gray circles represent helical regions. Coordinated zinc (Zn) and cysteine (C) residues arelabeled. Amino acids comprising the proximal box (P box) have lines drawn through them, whereas residues com-prising the dimerization box (D box) are darker gray. B, constructs of PR and JDP2 used in protein-protein interactionassays including PR DBD-CTE641, GST�JDP2, GST�JDP2 amino terminus (N) �basic domain, GST�JDP2 bZIP, GST�JDP2basic domain, and GST�JDP2 ZIP. C, PR DBD-CTE641 was incubated with free GST or the different GST�JDP2 fusionproteins immobilized to glutathione-Sepharose resins. After washing resins, bound protein was eluted anddetected by immunoblot assay with a PR DBD-specific antibody along with input PR DBD-CTE641.

FIGURE 2. NMR mapping of JDP2 interaction with the PR DBD-CTE. A, HSQC spectrum of PR DBD-CTE641. NMR spectra were recorded at 15 °C on a Varian600-MHz spectrometer using a triple-resonance (1H/13C/15N) pulsed field gradient cold probe. B, unlabeled JDP2 bZIP was titrated into PR DBD-CTE641, andmultiple HSQC experiments were recorded on the Varian 600-MHZ spectrometer. Overlay resonance assignments of PR DBD-CTE641 alone are shown in blue,and PR DBD-CTE641 plus JDP2 bZIP assignments are shown in red. Resonances that shift or disappear on the addition of JDP2 bZIP indicate sites on PRDBD-CTE641 that are perturbed by the binding of JDP2.

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beled PR DBD-CTE641, potential JDP2 binding sites were iden-tified as chemical shift differences between the free and boundforms of the protein. The relative chemical shift differences per

residue superimposed on a schematic of the PR DBD-CTE641(Fig. 3A) and a color map of the three-dimensional crystalstructure (21) (Fig. 3B) illustrate the regions of PR influenced by

FIGURE 3. Overlay of NMR chemical shift changes in the presence of JDP2 on the structure of the PR DBD-CTE. A, chemical shift changes between PRDBD-CTE641 alone and PR DBD-CTE641 with JDP2 bZIP were quantitated by [0.5(�NH2 � �N2/25)]1⁄2 (74). Larger differences indicate more of a shift change uponthe addition of JDP2 bZIP. Resonances with the largest perturbations by JDP2 are colored in red. D box, dimerization box. B, color-coded map of PR DBD-CTEcrystal structure (21) showing areas of chemical shift changes in the presence of JDP2 bZIP; red represents the greatest change, and gray represents the leastchange.

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JDP2 bZIP binding. The greatest changes in chemical shiftsoccurred in helix 1 of the core DBD, the dimerization box,and the CTE. In contrast, helices 2 and 2� of the core DBDshowed little change in backbone chemical shift, indicatingthat these regions do not interact with JDP2 bZIP or undergoa change in chemical environment as a result of JDP2 bZIPbinding.

NMR-guided Mutagenesis ShowsThat Specific Residues in the CTEAre Required for PR Interaction withJDP2—To examine the involvementof the PR DBD-CTE residues per-turbed in the NMR spectra for JDP2binding, we analyzed the effects ofspecific amino acid substitutions inthe PR DBD-CTE641 construct byGST pulldown assays. Amino acidsin the PR DBD-CTE641 that exhib-ited chemical shift changes in thepresence of JDP2 were substitutedonly if the change was predicted tonot disrupt global protein foldingbased on Ramachandran angles.Substitutions of core DBD residueshad little (G585A and R593Q) orminimal (K613Q) effect on JDP2binding, as did substitutions in twoCTE residues (G635A and G636A)that disappeared in the NMR spec-trum upon the addition of JDP2(Fig. 4A). These data suggest thatthese amino acids undergo a confor-mational change upon JDP2 bindingas opposed to representing residueswithin direct contact sites. How-ever, substitution of two other resi-dues in the CTE (R637A/K638A),which undergo chemical shiftchanges, abolished JDP2 binding(Fig. 4B). Truncation mutations ofthe CTE constructs were also exam-ined for their effects on binding toJDP2 (Fig. 5A). JDP2 bound withequal efficiency to CTE constructsdeleted from aa 650 to 648 and to641, whereas no binding was de-tected with further deletion of theCTE to aa 632 (Fig. 5B). These datacollectively indicate that residues inthe CTE closest to the core DBD (aa632–641) are most important forbinding JDP2 and that residues inthe core are dispensable.To further explore the role of the

CTE in binding JDP2, a syntheticpeptide corresponding to CTE se-quence aa 636–654 (Fig. 6A) wastested for its ability to compete for

JDP2 binding to PR. The CTE peptide inhibited the interactionbetween JDP2 bZIP andDBD-CTE650 in aGST pulldown assay,whereas a scrambled control peptide with the same amino acidcomposition had little effect (Fig. 6B). This result indicates thatthe CTE alone is sufficient for binding JDP2 and that a specificsequence is required. Taken together, these results indicate thatthe CTE plays a critical role in protein interaction with the

FIGURE 4. Specific residues in the CTE are required for JDP2-PR binding. A, single amino acid substitutionswere introduced into PR DBD-CTE641, and the effects of the mutations on binding to JDP2 were analyzed bypulldown assay. Varying amounts of WT or mutant PR DBD-CTE641 were incubated with either free GST orGST�JDP2�bZIP immobilized to glutathione-Sepharose resins. Bound protein was eluted after washing of beadsand detected along with input PR DBD-CTE641 by immunoblotting with a PR DBD-specific antibody. B, the twoamino acid substitutions indicated were introduced into PR DBD-CTE648 and compared with wild type (WT) PRDBD-CTE648 for binding to JDP2�bZIP by GST pulldown as in A above.

FIGURE 5. A subregion of the CTE is required for JDP2-PR binding. A, schematic of PR DBD-CTE truncationsused in GST pulldown assays. B, varying amounts of PR DBD-CTE constructs were incubated with either free GSTor GST�JDP2�bZIP immobilized to glutathione-Sepharose resins. Bound protein along with input PR DBD-CTEswas detected by immunoblot with a PR DBD-specific antibody.

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JDP2. Specifically, Arg637 and Lys638 are more crucial for JDP2binding, whereas Gly635 and Gly636, which had large changes inchemical shift, are predicted to be involved in the effects ofJDP2 on dynamics or conformation of the CTE.

JDP2 Interaction with the PR DBD-CTE Is Required forEnhancement of PR Activity in Cells—To determine whetherJDP2 interaction with the CTE observed in cell-free assays isimportant for its ability to enhance transcriptional activity ofPR, cell transfection assays were performed. Cos-1 cells were

co-transfected with a PRE-drivenluciferase reporter gene (PRE2-TATA-luc) and wild type PR-B orPR-B harboring two mutations inthe CTE residues (R637A/K638A)that disrupted binding to JDP2.Cos-1 cells have low levels of endog-enous JDP2, and as shown previ-ously (47, 48), ectopic expressionof JDP2 enhances progesterone-dependent PR-mediated transacti-vation of PRE2-TATA-luc (Fig.7B). Unexpectedly, the PR-B CTEmutant (R637A/K638A), in theabsence of ectopically expressedJDP2, had a stronger progesteroneinduction than wild-type PR, al-though the expressed protein lev-els were nearly identical (Fig. 7A).However, the enhancement ofreceptor-mediated transactivationof the luciferase reporter gene byJDP2was substantially reducedwith

FIGURE 6. The PR CTE alone is sufficient for binding JDP2-PR binding. A, sequences of synthetic peptidesused to compete for JDP2-PR binding. The CTE peptide is a 19-mer corresponding to aa 632– 656 of the PR CTE.sCTE (scrambled) is a 19-mer containing the same amino acid composition as the CTE except that the sequencewas randomly scrambled. B, PR DBD-CTE650 was incubated with either free GST or GST�JDP2�bZIP immobilizedto glutathione-Sepharose resins in the presence of varying amounts of either CTE or sCTE peptides. Boundprotein along with input PR DBD-CTE650 were eluted after washing resins and detected by immunoblottingwith a PR DBD-specific antibody.

FIGURE 7. Functional requirement of CTE for JDP2 enhancement of PR-mediated gene transcription in cells. A, mutations in CTE enhance progesterone-dependent transcription activity of PR in the absence of ectopically expressed JDP2. Cos-1 cells were transiently co-transfected with a PRE2-TATA-luciferasereporter gene and an expression plasmid for wild type PR-B or PR-B containing substitution mutations in the CTE (R637A/K638A). Cells were treated without(vehicle) or with progesterone (Prog) (10 nM) for 48 h and assayed for luciferase activity as described under “Experimental Procedures.” Results were calculatedas relative luciferase activity in vehicle versus progesterone treated cells and are average values � S.E. (error bars) from three independent experiments. Pairedtype 1 Student’s t test analysis was used to determine statistical significance for the difference of progesterone induction between PR-B and PR-B R637A/K638A;p � 0.05. The inset is an immunoblot of cell extracts used in luciferase assays with a PR-specific antibody (1294) to detect relative levels of WT PR-B and PR-BR637A/K638A mutant protein expression (PR-B is 118 kDa). B, mutations in CTE attenuated JDP2 enhancement of PR transcriptional activity. Cos-1 cells wereco-transfected as in A above with PR-B or PR-B CTE (R637A/K638A) and PRE-TATA-luc except in the absence (empty vector) or presence of varying amounts (25,50, 75 ng) of JDP2 expression plasmids. Results were calculated as -fold JDP2 stimulation of PR-mediated transactivation of luciferase reporter gene activity inthe presence of progesterone by setting relative luciferase activity of each receptor in the absence of transfected JDP2 to 1.0. Data represent average values �S.E. from three independent experiments. Paired type 1 Student’s t test analysis was used to determine statistical significance between the effect of JDP2 onPR-B and PR-B R637A/K638A; p � 0.05. C, swapping of TR for PR CTE eliminated JDP2 stimulation of PR transcriptional activity. Cos-1 cells were co-transfectedas in B except with PR-B or a PR�TR chimera consisting of a swap within full-length PR-B of the CTE from TR for PR CTE. Results were calculated as the -foldincrease of JDP2 stimulation in the presence by setting relative luciferase activity of each receptor in the absence of transfected JDP2 to 1.0. Data representaverage values � S.E. from three independent experiments. Paired type 1 Student’s t test analysis was used to determine statistical significance betweeneffects of JDP2 on PR-B and PR�TR chimera; p � 0.05.

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the PR-Bmutant (R637A/K638A) as compared with that of thewild-type PR (Fig. 7B).To further analyze the functional role of the CTE in cells, a

receptor chimera consisting of the CTE of PR-B, replaced withthe CTE of the thyroid receptor (TR), was transfected intoCos-1 cells in the absence and presence of increasing amountsof JDP2. Ectopic expression of JDP2 had no effect on progest-erone-induced transactivation mediated by the PR�TR chimeraas compared with wild type PR (Fig. 7C). Thus, the CTE of PRspecifically is required for functional response to JDP2 in cells.

DISCUSSION

The results presented here provide insights into the mecha-nismbywhich JDP2 interacts with PR to stimulate its transcrip-tional activity. The basic DNA binding domain is the minimalregion of JDP2 required for protein interaction with PR. Inter-action is not mediated directly through the leucine zipper.NMR spectroscopy coupled with the effects of specific aminoacid substitutions on PR activity have defined residues in a spe-cific region of the PR CTE asmost critical for JDP2 binding andfor functional response to JDP2 as a co-activator. Other resi-dues in the CTE and core DBD of PR that were perturbed in the

NMR spectra by JDP2, but were notrequired for JDP2 binding, undergoeither conformational changes oralteration in their chemical environ-ment due to other residues that areaffected structurally by binding toJDP2. JDP2-PR CTE interactionexhibits sequence specificity, asshown by the peptide competitionassay, and was necessary for JDP2co-activation of PR-mediated tran-scription in cells. These data, alongwith previous reports that the CTEis important for binding DNA andfor interactionwithHMGBproteins(28–31, 34), implicate theCTE as animportantmodulatory region of ste-roid receptors (Fig. 8).Of the residues in the CTE that

exhibited chemical shift changes inthe NMR spectra in the presence ofJDP2 (Fig. 2), only mutations inArg637 and Lys638 abolished JDP2binding; mutations in Gly635 orGly636 had no effect (Fig. 4). Basedon truncationmutations, sequencesof the CTE between aa 632 and 641that encompasses the residues wererequired for JDP2 binding, suggest-ing that this subregion constitutes aJDP2 binding site (Fig. 5) and thatmore than one site is required. Thefact that mutations of Gly635 andGly636 did not affect JDP2 bindingsuggests that the chemical environ-ment of these residues was per-

turbed by JDP2 binding to residues elsewhere (possibly Arg637and Lys638). Interestingly, the CTE sites implicated in directJDP2 binding are also involved in DNA binding (21), andanother subregion of the CTE (aa 641–651) was demonstratedpreviously to be required for binding the co-regulator protein,HMBG (31). These data suggest that the CTE is dynamic andcapable of binding protein or DNAwithin distinct and overlap-ping subregions (Fig. 8).Most steroid receptors have at least one glycine residue

between the Gly-Met (GM) boundary of the core DBD and theCTE that could provide the flexibility for the CTE to adoptdifferent conformations. In the recent crystal structure of a full-length PPAR-�-RXR-�heterodimer bound toDNA, theCTEofPPAR-� was observed to make contact with DNA, whereas theCTEof RXR-�makes dimer contacts with PPAR-�. The RXR-�CTE in particular lacks secondary structure, giving it flexibilitythat may be an adaptive feature to allow for more promiscuousprotein interactions and DNA binding (58). Flexibility of theCTE has also been demonstrated in high resolution crystalstructures of the orphan nuclear receptor ERR-2. The CTE ofERR-2 was unstructured in the absence of DNA and adoptedsecondary structure when bound to DNA (16, 22). The recently

FIGURE 8. Working hypothesis for the CTE as a multidimensional regulatory region of progesteronereceptor. The CTE is proposed to be a short, intrinsically disordered region of PR that is capable of dynamicinteractions with the minor groove of DNA or with multiple proteins including JDP2 or others such as HMGB.Distinct as well as overlapping residues in the CTE are important for interaction with different partners; aa632– 641 is most important for interaction with DNA and JDP2, whereas aa 642– 656 is more important forinteraction with HMGB (31). The CTE is capable of adopting distinct conformations dependent on the nature ofthe interacting partner, and JDP2-induced structural changes in the CTE are proposed to mediate couplingwith the disordered NTD of PR.

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reported computational methods of Wishart et al. (56) thatgenerate protein three-dimensional structure from NMRchemical shift assignments and protein sequence were used toanalyze the PR DBD-CTE641. This analysis indicated a changein conformation of the CTE in solution versus the crystal struc-ture of PR DBD-CTE641 bound to DNA. (supplemental Fig. 2).

Previous work from our laboratory demonstrated that JDP2functions as an NTD co-activator independent of AF2 in theLBD (47, 48). The current work suggests that the CTE plays acritical role for both JDP2 binding and co-activator function.Cell-based assays using CTE point mutations (R637A/K638A)showed a significant decrease in JDP2 enhancement of PRactivity. However, some effect of JDP2 still occurred, suggestingthat other residues in the CTE are also involved. The functionalrequirement of the CTE for JDP2 co-activation was also shownby analysis of a PR�TR chimera that contains the CTE of TR inplace of the PR CTE. In contrast to steroid receptor CTEs,which are flexible with little secondary structure, the CTEs ofTR and other non-steroid nuclear receptors are reported toexist as a stable�-helix (7).We previously showed that the CTEis a binding site for HMGB-1 and that it mediates the effects ofHMGB-1 to increaseDNAbinding of PR. TheTRCTEdoes notbind HMGB, and a PR�TR CTE chimera also failed to respondtoHMGB,whereas the reverseTR�PRCTE chimera, containingthe CTE of PR in place of TR, did respond to HMGB-1 (29, 30).These data suggest fundamental differences in the propertiesof TR and PR CTEs. Consistent with this conclusion, JDP2 hadno detectable effect on the transcriptional activity of a PR�TRCTE chimera, indicating that both the sequence and the struc-ture of the PR CTE are critical for PR co-activation by JDP2.How the short CTE can be a binding site for DNA and pro-

tein is an interesting and unanswered question. The CTEappears to be flexible and capable of interacting with multiplepartners in a dynamic and transient fashion. Based on theresults of the present work and earlier studies of the CTE inter-action with the minor groove of DNA and HMGB, we proposethat the CTE is a short IDP region capable of interacting withDNA and other proteins (Fig. 8). Many transcription factorshave IDP regions, indicating an important regulatory role forthese disordered regions (11–15). A recent model proposedthat intrinsic disorder in multidomain proteins can maximizethe ability to allosterically couple two domains and that theability to propagate the effects of binding partners is deter-mined by the energetic balancewithin the protein due to degen-erate requirements for coupling, rather than by a mechanicalpathway linking two protein domains (59). We have previouslyshown that the primary effect of JDP2 on the activity of PR isassociated with increased folding of the NTD (48). We proposethat the CTE is involved in coupling between the DBD andNTD domains through conformational changes in the CTEinduced by JDP2 binding.Structural and biochemical studies of bZIP proteins in com-

plexes with other proteins and DNA provide a working modelfor the structural basis for JDP2-PR interactions. Fos/Jun het-erodimer interaction with the transcription factor nuclear fac-tor of activated T-cells (NFAT) is mediated by contacts in thefork between the basic region and the coiled-coil region of theJun subunit, as well as the coiled-coil region of both Fos and Jun

(60, 61). However, in the crystal structure of ATF-2/cJun andIRF-3 bound to DNA, ATF-2 interacts with two loops, L1 andL3, of IRF-3 through only its basic region (62). The basic regionof bZIP proteins contains a distinct surface that directly con-tacts DNA and another involved in mediating interaction withco-activators. For example, the selectivity of Chameau andMBF1 histone acetylases for bZIP proteins is mediated by resi-dues in the basic region that lie on the opposite surface from theresidues that contact DNA (63).In addition to JDP2, other co-activators that bind to the

DBDs of steroid receptors have been described. The smallnuclear RING finger protein (SNURF) binds to theDBDof hor-mone activated androgen receptor (AR), ER, and PR to enhancesteroid receptor-dependent transcription and has been sug-gested to act as a bridging factor between steroid receptors andother transcription factors (64–66). GT198 interacts withmany NRs through their DBDs to enhance transcription,including ER� and -�, TR�1, androgen receptor (AR), glu-cocorticoid receptor (GR), and PR (67). The co-activator andhistone acetylase PCAF (p300/CREB-binding protein-associ-ated factor) interacts, independently of p300/CREB-bindingprotein, with anRXR-RAR (retinoic acid receptor) heterodimerthrough the DBD of either receptor (68). The ER�-associatedprotein pp32 has been shown to interact with the ER DBD in ahormone-independent manner and to increase ER�-ERE com-plex formation but acts to repress ER�-mediated transcription(69). pp32 was also shown to decrease the acetylation of ER�both in vitro and in cells, whichmay play a role in its co-repres-sor activity. More recently, proliferating cell nuclear antigen(PCNA) was shown to interact with the DBD of ER� and toenhance receptor-DNA interaction in vitro, leading toincreased basal transcription of endogenous estrogen-respon-sive genes. Interestingly, it was found that proliferating cellnuclear antigen interacts with the CTE region of the ER� DBD(70). Additionally, DNA has been reported to act as ligand andto induce conformational changes in the DBD and/or tran-scription activation surfaces of NRs, thereby influencing co-regulatory protein interactions and NR function (71–73).Despite the number of DBD-interacting co-factors reported,there has been little work to define interaction sites within theDBD or mechanisms of co-activation. Because most of the sur-faces of the core DBD are involved in interactions with DNA ordimerization, it is most likely that the unstructured CTE is aprotein interaction site for many other co-regulators.

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EdwardsKrista K. Hill, Sarah C. Roemer, David N. M. Jones, Mair E. A. Churchill and Dean P.

DomainInteraction with Residues in the Carboxyl-terminal Extension of the DNA Binding

A Progesterone Receptor Co-activator (JDP2) Mediates Activity through

doi: 10.1074/jbc.M109.003244 originally published online June 24, 20092009, 284:24415-24424.J. Biol. Chem. 

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