the juvenilehormonereceptorcandidates methoprene-tolerant ... · met and gce proteins and explore...

The Drosophila Juvenile Hormone Receptor CandidatesMethoprene-tolerant (MET) and Germ Cell-expressed (GCE)Utilize a Conserved LIXXL Motif to Bind the FTZ-F1 NuclearReceptor*□S

Received for publication, November 23, 2011, and in revised form, January 5, 2012 Published, JBC Papers in Press, January 16, 2012, DOI 10.1074/jbc.M111.327254

Travis J. Bernardo‡ and Edward B. Dubrovsky‡§1

From the ‡Department of Biology and §Center for Cancer, Genetic Diseases, and Gene Regulation, Fordham University,Bronx, New York 10458

Background: JH signaling involves interactions between FTZ-F1 and candidate JH receptors MET and GCE.Results:Mutation of FTZ-F1 AF2 or LIXXL MET/GCE sequence disrupts interaction between the proteins.Conclusion: NR box-AF2 binding underlies FTZ-F1�MET and FTZ-F1�GCE heterodimer formation.Significance:Dissecting the interaction between FTZ-F1 andMET, GCE is critical to understanding the molecular basis of JHsignaling.

Juvenile hormone (JH) has been implicated in many develop-mental processes in holometabolous insects, but its mechanismof signaling remains controversial. We previously found that inDrosophila Schneider 2 cells, the nuclear receptor FTZ-F1 isrequired for activation of the E75A gene by JH.Here, we utilizedinsect two-hybrid assays to show that FTZ-F1 interacts with twoJH receptor candidates, the bHLH-PAS paralogsMET andGCE,in a JH-dependent manner. These interactions are severelyreducedwhenhelix 12of theFTZ-F1 activation function2 (AF2)is removed, implicating AF2 as an interacting site. Throughhomology modeling, we found that MET and GCE possess aC-terminal �-helix featuring a conservedmotif LIXXL that rep-resents a novel nuclear receptor (NR) box. Docking simulationssupported by two-hybrid experiments revealed that FTZ-F1�MET and FTZ-F1�GCE heterodimer formation involves atypical NR box-AF2 interaction but does not require the canon-ical charge clamp residues of FTZ-F1 and relies primarily onhydrophobic contacts, including a unique interactionwith helix4.Moreover, we identified paralog-specific features, including asecondary interaction site found only inMET.Our findings sug-gest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1.

Juvenile hormone (JH)2 and 20-hydroxyecdysone (ecdysone)have prominent roles in regulating insect development. In the

larvae of holometabolous insects, high JH titer forces ecdysoneto elicit molting, whereas a drop in JH during the final larvalinstar allows ecdysone to trigger entry into prepupal develop-ment (1). The ability of JH to delay metamorphosis has beenobserved directly in insects, such as the silkwormBombyxmori,where removal of endogenous JH by ectopic JH esterase resultsin premature pupariation (2) and in the red flour beetle Tribo-lium castaneum, where exogenous JH preventsmetamorphosisthrough the production of additional larval instars (3, 4).There is accumulating evidence that the antimetamorphic

function of JH involves MET (methoprene-tolerant), a basic-helix-loop-helix Per-AhR/Arnt-Sim (bHLH-PAS) protein andcandidate JH receptor with homologs identified in all holo-metabola whose genomes have been sequenced. TheTriboliumand Drosophila homologs of MET bind JH with nanomolaraffinity when produced in vitro (5, 6). In Tribolium MET isrequired to prevent premature pupariation in larvae (3, 4) andacts upstream of the antimetamorphic gene Kr-h1, a target ofJH activation (7). In Drosophila, the role of MET as a mediatorof JH action is less clear. It is required in vivo for JH-dependentdevelopment of the adult eyes and CNS optic lobes (8) and forthe lethal “methoprene syndrome” response to ectopic hor-mone (9). However, despite the fact that JH deficiency is lethal,Met null mutant flies are viable. In contrast to insects thatdependonMET for survival, drosophilids possess aMetparalogencoded by the gce (germ cell-expressed) gene (10). GCE is alsoa bHLH-PAS protein and is capable of binding JH in vitro (5).LikeMet, gce null mutants are viable and insensitive to ectopicJH (11). Although neithermutation is lethal, mutants missingboth paralogs die as prepupae, suggesting that MET andGCE have some redundant functions in vivo. However, theparalogs are not completely redundant because their abilityto substitute for one another in JH-dependent processes istissue-specific (9). Recently, we found that JH activation ofthe nuclear receptor gene E75A in S2 cells requires GCE but

* This work was supported by National Science Foundation Grant 0653567(to E. B. D.) and a Fordham University Graduate School of Arts and Sciencesresearch fellowship (to T. J. B.).

□S This article contains supplemental Figs. S1–S3, Tables S1 and S2, and modelfiles MET(bHLH).pdb, MET(PAS).pdb, GCE(bHLH).pdb, and GCE(PAS).pdb.

1 To whom correspondence should be addressed: 441 E. Fordham Rd., Ford-ham University, Bronx, NY 10458. Tel.: 718-817-3660; Fax: 718-817-3645;E-mail: [email protected].

2 The abbreviations used are: JH, juvenile hormone; ecdysone, 20-hy-droxyecdysone; bHLH, basic-helix-loop-helix; PAS, Per-AhR/Arnt-Sim; PAC,PAS-associated C-terminal motif; QR, glutamine-rich; DBD, DNA-bindingdomain; LBD, ligand-binding domain; AF1 and AF2, activation function 1and 2, respectively; S2, Schneider 2; SRC, steroid receptor coactivator; NR,

nuclear receptor; AIR, ambiguous interaction restraint; PDB, Protein DataBank.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 10, pp. 7821–7833, March 2, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7821

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not MET, indicating that GCE possesses paralog-specificregulatory functions (12). Despite the emerging evidencethat bothMET andGCE are involved in JH signaling, it is stillnot clear whether MET, GCE, or both proteins act as bonafide JH receptors in flies.The structure of MET and GCE conforms with their pro-

posed role in regulating transcription. The bHLH is a DNA-binding domain found in many transcription factors (13). PASdomains often bind ligands and can function as signal sensorsfor light, oxygen, redox potential, metabolites, and xenobioticcompounds (14). They can also mediate protein-protein inter-actions. For example, mosquito MET utilizes its PAS domainsto interact with the bHLH-PAS partner FISC; together theybind a JH-responsive sequence and activate transcription of theearly trypsin gene (15). The location of the bHLH and PASdomains in MET and GCE is typical of the bHLH-PAS family;the bHLH is at the N terminus and is followed by two PASmotifs, PAS A and PAS B, and a single PAS-associated C-ter-minal (PAC) motif. PAS motifs are a characteristic feature ofPAS domains, and in MET and GCE they share considerablesequence identity with other PAS proteins (16, 17). A completePAS domain cannot always be identified by primary structure,however, because the corresponding sequences often have alimited degree of identity outside of the PAS motif (18). Thisappears to be the case for MET and GCE, where the tertiarystructure of the PAS domains has not yet been determined.Another common feature of bHLH-PAS proteins is the pres-ence of acidic, proline/serine (P/S), and glutamine-rich (QR)sequences in the C terminus that can serve as transactivationdomains (19). However, the C-terminal regions of MET andGCE have not yet been functionally characterized.Recently, we found that in addition to GCE, JH activation of

E75A requires the orphan nuclear receptor FTZ-F1 (12). Theftz-f1 gene encodes two isoforms, �ftz-f1 and �ftz-f1, whoseproducts share an identical DNA-binding (DBD) and ligand-binding (LBD) domains but have distinct N-terminal domainsresulting fromalternative promoter usage (Fig. 1A). The LBDofFTZ-F1 has a typical structure consisting of 12�-helices and ananti-parallel �-sheet; it forms a coactivator interaction surfacereferred to as activation function 2 (AF2) from helices H3, H3�,H4, and H12 (20, 21). In contrast to ligand-dependent AF2, theAF2 of FTZ-F1 is stabilized in an active conformation by H6,which serves as a pseudoligand and may allow FTZ-F1 to func-tion as a ligand-independent activator (21). The presence of aconstitutively active AF2 suggests that FTZ-F1 could act pri-marily through protein-protein interactions, and our findingthat FTZ-F1 interacts with both MET and GCE (12) raises thepossibility that FTZ-F1 mediates JH action indirectly throughthe formation of FTZ-F1�MET or FTZ-F1�GCE heterodimers.Previous examples of interactions between bHLH-PAS pro-teins and nuclear receptors have come from the steroid recep-tor coactivator (SRC) family of proteins. A centrally locateddomain in these proteins contains three copies of a motif,LXXLL, termed the nuclear receptor (NR) box, which adopts an�-helical conformation and binds to a hydrophobic grooveformed by the AF2 (20, 22). For the SRC coactivators, each NRbox is critical to interaction with a different subset of nuclearreceptors. The N-terminal bHLH and PAS domains of SRC

proteins are dispensable to their interactionwith nuclear recep-tors (23–25), whereas a QR region in the C terminus is used as asecond interaction site through a poorly understood mecha-nism (26–29). In addition to the SRC family, NR boxes havebeen identified in a variety of other coregulators, including thehomeodomain protein FTZ, which utilizes an NR box to inter-actwith theAF2 of FTZ-F1 (21, 30, 31). No functionalNRboxeshave yet been identified in MET or GCE, so the mechanism oftheir interaction with FTZ-F1 remains unresolved.Here, we define the secondary and tertiary structure of the

MET and GCE proteins and explore the molecular basis of theFTZ-F1�MET and FTZ-F1�GCE interactions. We demonstratethat bothMET and GCE interact JH-dependently with FTZ-F1AF2, utilizing a novel NR box with a unique and paralog-dis-tinct mechanism.

EXPERIMENTAL PROCEDURES

Cell Culture, Cell Transfection, and RNAi—Drosophila S2cells were cultured in Schneider’s medium (Invitrogen) supple-mented with 10% fetal bovine serum (FBS) at 25 °C. JH III (anatural JH compound; Sigma) and methoprene (a synthetic JHanalog) were dissolved in ethanol, and 1� 10�1 M stock solutionswere kept at�20 °C. Hormones were added to a 1� 10�6 M finalconcentration. Control cells were treated with an equal vol-ume of ethanol. Transient transfection of expression plas-mids and RNAi treatment of S2 cells were performed asdescribed previously (12, 32).Plasmids and Mutagenesis—The ORFs for �FTZ-F1 and

comFTZ-F1 (residues 175–803, �-isoform numbering) wereamplified by PCR using �ftz-f1 cDNA acquired from the Dro-sophila Genomics Resource Center (EST clone RE02257) andcloned into the pMt/V5-His plasmid (Invitrogen) using primerswith flanking restriction sites. For expression of �FTZ-F1, the�-specific coding region was amplified from S2 genomic DNAand cloned into the comFTZ-F1 expression plasmid, producing�FTZ-F1 ORF with a two-residue linker. Protein expressionwas confirmed by Western blot with anti-V5 antibodies(1:10,000 dilution). To produce double-strandedRNA (dsRNA)for RNAi knockdown of �ftz-f1, a DNA fragment containing�-specific protein coding sequences was amplified by PCR andcloned in both orientations into the pGEM-T Easy plasmid(Promega) using the following primers: 5�-TGC TCT AGAATG ACA CTA ATG GGC ACT GC-3� and 5�-TGC TCTAGA TGC TAG TGT GGT TGC TGT TG-3�. Plasmids forthe insect two-hybrid experiment (pIE2-(p65)AD, pIE2-(GAL4)DBD, and pUAS-Luc) were a gift from Dr. T. Kusakabe(KyushuUniversity).MET-DBD, GCE-DBD, and FTZ-F1-ADfusion constructs were generated using the pENTR3C plas-mid and LR Clonase II kit (Invitrogen). The constitutiveexpression plasmid pAc5.1-LacZ-V5 (Invitrogen) was usedin two-hybrid experiments to measure transfection effi-ciency. Point mutations were introduced using theQuikChange II site-directed mutagenesis kit (Stratagene)according to the manufacturer’s instructions. All expressionplasmids were sequenced (Genewiz) to confirm the ORFs.Primer sequences are available upon request.

NR Box Motif in MET and GCE Mediates Interaction with FTZ-F1

7822 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

RNA Extraction, Northern Hybridization, and QuantitativeRT-PCR—Extraction of RNA,Northern blot hybridization, andquantitative RT-PCR were performed as described previously(12). Primer sequences are available upon request.Luciferase and �-Galactosidase Reporter Assays—Assays

were performed as described (12). Briefly, standardized proteinextracts were used to measure luciferase using the LuciferaseAssay System (Promega), and relative light units were averagedover 10 s for each sample.�-Galatosidase activitywasmeasuredwith the �-Galactosidase Assay System (Promega), withabsorbance at A420 recorded every 60 s for 10 min. Luciferaseactivity was normalized to �-galactosidase activity for eachsample.Homology Modeling—Models of MET and GCE tertiary

structure were built using Phyre2 (33). PSI-BLAST alignmentsof MET and GCE amino acid sequences were used to identifyremote homologs; each identified 1000 high confidence (E �0.001) matches primarily within the region containing the PASmotifs. The sequence identity to remote homologs was limited(ranging from 7 to 14%), indicative of highly informative align-ments (33). Secondary structure was predicted using threeindependent algorithms (Psi-Pred, SSPro, and JNet), and disor-dered regions were predicted using Disopred. The structuralpredictions and PSI-BLAST profile were used to search empir-ical structures from the Structural Classification of Proteinsdatabase and the Protein Data Bank (PDB). Among the topscoringmatches, nearly all of themwere either helix-loop-helixor PAS folds. Models for MET and GCE were built usingmurine MyoD for the bHLH domains and PER (DrosophilaPeriod) for the PAS domains.To assess the quality of Phyre2models, Z-scores were gener-

ated using QMEAN, which compares models to a reference setof empirical structures whose average Z-score is zero (34).Z-Scores for the bHLH domains of MET (�0.41) and GCE(�0.25) indicate models that are comparable with experimen-tally derived structures, whereas Z-scores for the PAS domainsof MET (�1.91) and GCE (�2.85) indicate medium qualitymodels. Local (i.e. per residue) QMEAN scoring of the PASdomain models found poor quality modeling in the regionsdirectly adjacent to PAS loops and in the region linking the twoPAS folds, whereas the�-strands and�-helices of the PAS foldsare of higher quality. Final model illustrations were producedusing PyMOL.Identification of Conserved MET and GCE Residues—Multi-

ple sequence alignment of MET and GCE homologs was per-formed using ClustalW (35). After alignment, the percentage ofhomologous (i.e. identical or similar) residues among all pro-teins was calculated for each conserved sequence block identi-fied in the alignment. The complete set of conserved sequenceblocks identified among distant MET and GCE homologs isshown in supplemental Fig. S2; for alignment of C-terminalMET and GCE sequences, see supplemental Fig. S3.Simulated Docking of Protein Complexes—Docking models

of FTZ-F1�MET and FTZ-F1�GCE complexes were generatedwith HADDOCK (36). The three-dimensional structure of theFTZ-F1 LBD is publicly available (PDB entry 2XHS). 9-residueNR box peptide structures for MET (LYLIENLQK, residues552–560) and GCE (LRLIQNLQK, residues 416–424) were

generated de novo by manually mutating the side chains of the9-residue FTZ NR box peptide from the FTZ-F1 crystal struc-ture. Parallel docking was performed using the FTZ NR box(STLRALLTN, residues 107–115), whose binding to FTZ-F1has been determined empirically (21). Docking by HADDOCKis driven by predictions of likely residues involved in complexformation (ambiguous interaction restraints (AIRs)); thesemaybe active (interacting residue) or passive (solvent-accessibleneighbor of interacting residue). AIRs for FTZ-F1 were gener-ated using CPORT (37), and AIRs forMET and GCE were cho-sen based on experimental data (supplemental Table S1). Foreach docking experiment, 10,000 rigid body simulations wereperformed, followed by semiflexible simulated annealing, waterrefinement, and clustering of the top 400 simulations based onstructural similarity; clusters were then assigned energy scores.The largest cluster of docking solutions in each experiment hadthe most favorable (i.e. lowest) energy score, and the scoreswere similar when either MET, GCE, or FTZ peptides weredocked (supplemental Table S2). As expected, the structure ofthe docked FTZpeptidewas similar to that of FTZ in the crystalstructure (root mean square deviation of peptide from the low-est energy structure versus the crystal structure was 1.00 Å).Following docking simulations by HADDOCK, the lowestenergy structures were refined using Rosetta FlexPepDock,which improves the accuracy of docking by allowing for flexi-bility in the backbone of short peptides (38). MolProbity (39)was used to identify hydrogen bonds and non-bonded contacts.Final model illustrations were produced in PyMOL.

RESULTS

Both Isoforms of FTZ-F1 Can Mediate JH Activation—ftz-f1encodes two transcript isoforms, �ftz-f1 and �ftz-f1, which aredifferentially expressed during development (40, 41). To deter-mine which of these transcripts is present in S2 cells, probestargeting the isoform-specific 5� and common 3� regions (Fig.1A) were used to measure ftz-f1 gene expression by Northernblot hybridization. RNA samples were collected from S2 cellsbefore and after treatment with ecdysone and from prepupalstage larvae. The �-probe detected specific transcripts in S2cells (5.4 and 6.2 kb), whose expressionwas repressible by ecdy-sone, whereas the �-probe detected transcripts (4.8 and 5.6 kb)only in prepupal larvae (Fig. 1B). The common probe facilitateddirect comparison of transcripts from S2 cells and prepupae;the longer transcripts found in S2 cells compared with prepu-pae are consistent with the longer 5� region of �ftz-f1. Theseresults demonstrate that �ftz-f1 is the predominant transcriptin S2 cells, indicating that �FTZ-F1 protein is responsible formediating JH activation of E75A in this cell line.To determine whether the function of FTZ-F1 in JH signal-

ing is limited to �FTZ-F1, we studied JH inducibility of E75A incells that ectopically express high levels of �FTZ-F1, �FTZ-F1,or an N-terminally truncated FTZ-F1 containing only thedomains common to both isoforms (comFTZ-F1). Cells weretransfected with empty plasmid or plasmid expressing one ofthe FTZ-F1 proteins and then treated with solvent or the JHanalog methoprene for 1 h. In each case, the amount of proteinexpressed was comparable (Fig. 2A). In the absence of ectopicprotein, E75A is induced�3-fold after 1 h of JH treatment (Fig.







2B). When either �FTZ-F1 or �FTZ-F1 is present, E75A tran-scription ismodestly but significantly (p� 0.05) enhanced bothin the absence and presence of JH, resulting in higher overallexpression but a similar 3-fold level of JH induction (Fig. 2B).Removal of the isoform-specific region of FTZ-F1 that typicallyharbors ligand-independent activation function 1 (AF1) did notaffect E75A transcription in the absence of JH but modestlyenhanced transcription when hormone was present, resultingin 4-fold JH induction of E75A. These results indicate thatregardless of whether the hormone is present or not, both�FTZ-F1 and �FTZ-F1 potentiate E75A transcription, but thedomains shared between both isoforms are sufficient to poten-tiate JH-dependent induction.To determine conclusively whether both isoforms are func-

tionally equivalent in mediating JH signaling, we carried out anRNAi rescue experiment by utilizing the distinct 5� codingregion of the �ftz-f1 transcript. First, �ftz-f1 expression wasknocked down in S2 cells using dsRNA targeting the 5� region

specific to the � isoform. Cells were then transfected withempty plasmid or the �ftz-f1-encoding plasmid and treated for1 h with methoprene. RNAi treatment reduced endogenous�ftz-f1 expression by about 70% (data not shown) and resultedin a strong and significant (p � 0.05) reduction of E75A JH-de-pendent transcription (Fig. 2C); this result confirms that the �isoform is responsible for JH activation in this cell line. When�FTZ-F1 is expressed in an �ftz-f1 RNAi background, JH acti-vation of E75A is restored. These results demonstrate that both�FTZ-F1 and �FTZ-F1 can mediate the JH-dependent activa-tion of E75A.The AF2 of FTZ-F1 Is Required for JH-dependent Interaction

with MET and GCE—We previously found that FTZ-F1 inter-actswith the bHLH-PAS transcription factorsMETandGCE in

FIGURE 1. Isoform-specific expression of ftz-f1 in the S2 cell line. A, struc-tural organization of the ftz-f1 gene. Black bars, exons; arrows, promoters forthe � and � isoforms. Each isoform possesses a unique coding sequence atthe 5� end. Rectangles above the exons indicate probes specific for the �(white) and � (gray) isoforms as well as a common probe (striped). Alternativepolyadenylation sites are indicated by vertical arrows. Shown at the top is thesize of ftz-f1 in kilobases. B, total RNA was isolated from S2 cells cultured in thepresence of 1 � 10�6

M ecdysone for the time period indicated above eachlane and from prepupae (PP). RNA samples were analyzed by Northern blothybridization with radioactive probes for �-specific, �-specific, or commonexons. Transcript sizes are indicated on the right. rp49 expression was used asa loading control.

FIGURE 2. Both FTZ-F1 isoforms mediate JH-dependent activation ofE75A. A, expression of ectopic FTZ-F1 proteins used in B and C was confirmedby Western blot hybridization (IB) using anti-V5 antibody, with tubulin as acontrol for loading. B, S2 cells were transfected with empty plasmid or expres-sion plasmid encoding �FTZ-F1, �FTZ-F1, or common domains of FTZ-F1 asindicated (x axis) and then treated with ethanol solvent (light bars) or 1 � 10�6

M

methoprene (dark bars) for 1 h. Total RNA was extracted, and E75A expressionwas measured as -fold abundance against rp49 by quantitative RT-PCR (yaxis). Bars, mean � S.D. from three independent experiments. *, significant(p � 0.05) JH-dependent increase in E75A expression; **, significant JH-inde-pendent E75A activation. C, S2 cells were incubated for 48 h with double-stranded RNA targeting the 5� region of the �ftz-f1 transcript (�ftz-f1 dsRNA),transfected with empty plasmid or �ftz-f1-encoding plasmid as indicated onthe x axis, and treated with ethanol (light bars) or 1 � 10�6

M methoprene(dark bars) for 1 h. Expression of E75A relative to rp49 was measured as in B.Bars, mean � S.D. from three independent experiments. *, significant (p �0.05) change in E75A expression.



GST pull-down assays (12). Because nuclear receptors typicallyutilize the AF2 function of their LBD to interact with coregula-tors (20), we reasoned that FTZ-F1 may use its AF2 to formheterodimers with MET and GCE. To test this possibility, weemployed a two-hybrid system designed to quantitativelymeasure protein-protein interactions in cultured insect cells(42). GAL4-DBD fusions of MET or GCE were coexpressed inS2 cells with p65-AD fusions of �FTZ-F1, �FTZ-F1, or mutantFTZ-F1 with a 15-residue C-terminal deletion. The truncatedprotein (�FTZ-F1�H12) lacks the helix H12, which is critical tothe AF2 function of nuclear receptors (20).WhenMET or GCEare expressed alone, little or no reporter activity is seen whencells are treated with methoprene (Fig. 3). However, wheneither MET or GCE is coexpressed with �FTZ-F1, substantialJH-dependent reporter activity is evident (4.3- and 3.0-fold,respectively). Similar JH-dependent activity is observed wheneither MET or GCE is coexpressed with �FTZ-F1 (4.7- and3.9-fold, respectively). These results demonstrate that in thetwo-hybrid system, MET and GCE interact with both isoformsof FTZ-F1, and these interactions are JH-dependent. WhenH12 is removed from FTZ-F1, the JH-dependent interaction ofMET with the truncated FTZ-F1 is reduced significantly (p �0.05), but the proteins still retain a notable level of interaction(Fig. 3A). For GCE, the interaction with truncated FTZ-F1 isnearly abolished (Fig. 3B). These results indicate that FTZ-F1requires an intactAF2 to bindMETandGCE,with each paralogdisplaying distinct requirements for this motif.Characterization of MET and GCE Structure Identifies

Potential FTZ-F1 Interaction Sites—It has been well docu-mented that the AF2 of nuclear receptors forms a groove thatinteracts with �-helical NR box peptides containing thesequence LXXLL (20). In addition to this motif, other �-helicalstructures resembling LXXLL (e.g. LXXIL, LLXXL, and FXXLL)have been identified as functional NR boxes (43–45). There isonly one LXXLL motif, situated N-terminal to PAS A in MET,that potentially represents a canonical NR box, LMQLL. How-ever, this motif is not found in GCE or any other insect homo-log, and its deletion fromMEThas no impact on the interactionwith FTZ-F1. In order to identify any �-helices in MET andGCE containing non-conventional NR box motifs, we charac-terized the structure of these proteins with Phyre2 (33). Phyre2generates models of tertiary structure by homology to empiri-cally derived templates, and when a template is not available, itprovides information about secondary structures.MET and GCE are multidomain proteins, containing both

bHLH and PAS, and as a result Phyre2 identified three-dimen-sional models for each of these domains. Using the bHLH tran-scription factor MyoD (PDB entry 1MDY) as a template, mod-els were built for MET and GCE (supplemental Fig. S1) whosebHLH corresponds to residues 38–90 and 9–58, respectively.The domain consists of a long �-helix containing the N-termi-nal basic region (MET38–63, GCE 9–34), a loop (MET64–73,GCE 35–44), and a second shorter �-helix (MET 74–90, GCE45–58). The structure of the MET and GCE bHLH is typical ofthis domain (46), and as expected, no putative NR boxes wereidentified in this region.For modeling of the PAS region,Drosophila PER (PDB entry

1WA9) was used as a template. PER is a founding member of

the PAS family and, similar to MET and GCE, possesses twotandem PAS motifs (47). MET and GCE both adopt two PASfolds (designated 1 and 2) that correspond to residues 134–506and 83–371, respectively (Fig. 4A). Each fold consists of a five-strand antiparallel �-sheet flanked on one side by three or four�-helices (i.e. a canonical PAS fold) (18). PAS Fold 1 is com-posed of PAS A and three subsequent sequence blocks (I–III)(Fig. 4B). In this first fold, there are two flexible loops that varyin size betweenMET andGCE (Table 1), directly following PAS

FIGURE 3. JH-dependent interaction of MET and GCE with FTZ-F1 requireshelix 12. A and B, S2 cells were transfected with 4� UAS-TATA-Luc reporterconstruct, along with expression vectors for GAL4 and p65 fused to FTZ-F1,MET, or GCE as indicated (y axis). After transfection, cells were treated withethanol (light gray) or 5.0 � 10�6

M methoprene (dark gray) for 24 h. Luciferaseactivity was normalized to �-galactosidase activity from a constitutivereporter (x axis). Interactions of MET (A) and GCE (B) were measured with�FTZ-F1, �FTZ-F1, and C-terminally truncated �FTZ-F1�H12. Data are shownas the mean � S.D. from at least three independent experiments. *, significant(p � 0.05) difference in JH-dependent reporter activity between �FTZ-F1 and�FTZ-F1�H12. C, equal expression of fusion proteins was confirmed by West-ern blot (IB) using anti-FLAG or anti-HA antibody as indicated, with anti-tubu-lin antibodies used as a loading control. *, a nonspecific band.




A and between blocks II and III. PAS Fold 2 is composed of PASB and PAC, with no intervening loops. Inspection of individualsecondary structures (Table 1) reveals that each �-helix in thePAS region is part of a PAS fold. Given that typical NR boxes,such as those found in the bHLH-PAS-containing SRC coacti-vators, are always isolated from other domains (22), the regionofMETandGCEcontaining the PAS folds is unlikely to containan NR box.No template was found for the remaining C-terminal resi-

dues of MET (residues 507–716) and GCE (residues 373–689).Analysis of secondary structures in this region reveals thatalthough most of the C terminus consists of paralog-specificfeatures, there are two sequence blocks (IV and V) with similar�-helical structure and position in both proteins (Fig. 4B).Block IV (MET 507–524, GCE 373–390) is an 18-residue �-he-lix immediately following PAC. Block V (MET 547–558, GCE411–422) is a 12-residue �-helix following block IV, flanked oneither side by disordered regions. These helices, designated�IVand �V (Table 1), are distinct from other protein structures,and we considered them as candidate sites for an NR box.

Identification of Novel NR Box Motif in C Terminus of METand GCE—To assess the functional relevance of �IV and �V, amultiple sequence alignment was generated based on theassumption that functionally important residues are conserved.MET orGCE homologs have been identified in all holometabo-lous insects with sequenced genomes and in several cases havebeen implicated in JH signaling (3, 8, 12, 15). We thereforealigned MET and GCE from Drosophila melanogaster tohomologs from two insects of the order Diptera, a distant rela-tive within the Drosophila genus (Drosophila grimshawi) and amosquito species (Aedes aegypti).We also included in the align-ment species from the distant orders Coleoptera (Triboliumcastaneum) andHymenoptera (Nasonia vitripennis). Together,these insects provide an evolutionary distance ranging from 60to 300 million years (48, 49). The alignment revealed that �IVand �V exhibit sequence homology (61 and 58%, respectively)similar to that displayed by other conserved domains (Table 1and supplemental Fig. S2). These two �-helices (�IV and �V)are thus well conserved, suggesting they provide some criticalfunction in MET and GCE.

FIGURE 4. Structure of MET and GCE. A, three-dimensional structure of the two PAS folds from MET comprising residues 134 –506, as predicted by Phyre2.�-Helices and �-strands are colored according to the annotated motifs PAS A (green), PAS B (blue), and PAC (yellow) and sequence blocks I–III (red). Each PAS foldcontains five �-strands (�A--�E and �A�–�E�) and three or four �-helices (�A–�C and �A�–�C�). The fourth �-helix present only in PAS Fold 2 is denoted by anasterisk. A similar model was observed for GCE (not shown). B, schematic illustration of MET and GCE protein structures. Shown are the bHLH domain; the PASA, PAS B, and PAC motifs; sequence blocks I–V; and the acidic (Ac), proline/serine (P/S), and glutamine (QR) regions described in this paper. An arrowheadindicates the location of an LXXLL sequence in MET; the vertical arrows indicate the novel LIXXL NR boxes in MET and GCE identified in this report. Numbers atthe bottom of each schematic designate residue position.




The sequence alignments of �IV and �V were inspected forpotential NR box motifs (Fig. 5A). Intriguingly, �V possesses amotif whose sequence, L(I/L)XXL, is reminiscent of the NRbox. In contrast to the LXXLL sequence found only in MET(Fig. 4B, arrowhead), this LIXXL motif is present in both METand GCE (Fig. 4B, vertical arrow) and is conserved in otherinsects (Fig. 5A). Becausemutation of either of the outer leucineresidues of an NR box is sufficient to completely abrogate itsfunction (50), a conserved leucine at the �1 position wasmutated to alanine in MET and GCE (L554A and L418A,respectively), and their interaction with FTZ-F1 was measuredin the two-hybrid system. For both METL554A (Fig. 5B) andGCEL418A (Fig. 5C), we observed a significant (p � 0.05) reduc-tion in the interaction with FTZ-F1 compared with the wildtype proteins. By contrast, deletion of the �4 and �5 residuesfrom the LXXLL motif of MET had no significant effect on theinteraction with FTZ-F1 (Fig. 5B). The reduced ability ofMETL554A and GCEL418A to interact with FTZ-F1 suggestedthat the conserved motif in �V is a novel NR box used to inter-act with AF2.We noticed that mutation of the NR box did not fully abro-

gate the FTZ-F1�MET and FTZ-F1�GCE interactions. This wasespecially evident for MET, which retained roughly 50% of itsinteraction in the absence of theNR box (Fig. 5B), similar to theresidual interaction of MET with H12-deficient FTZ-F1 (Fig.3A). To identify other possible interacting sites, we inspectedresidues C-terminal to the NR box. Although this region ispoorly conserved among distant homologs (Table 1) we foundseveral regions conserved in the Drosophila genus, including aQR region (Fig. 4B and supplemental Fig. S3), which is used as asecondaryNR interaction site by SRC familymembers (26–29).Alignment of the MET and GCE QR regions revealed a shortblock of common glutamine/glutamate residues (Fig. 5A).Mutation of amino acids within this QR stretch by itself doesnot significantly disrupt the FTZ-F1�MET interaction, butwhen the NR box is also mutated, the interaction betweenFTZ-F1 and MET is completely abolished (Fig. 5B). A very dif-

ferent result was seen for GCE, because mutation in the QRregion had no disruptive effect whatsoever on the interactionwith FTZ-F1 (Fig. 5C). These results indicate that, in additionto the NR box, MET utilizes a secondary interaction site in theC-terminal QR.Unique NR Box-AF2 Complex Underlies FTZ-F1�MET and

FTZ-F1�GCE Interactions—Because the NR box in MET andGCE consists of a novel sequence (LIXXL), we were interestedin investigating themolecular basis of its interaction with FTZ-F1. We therefore modeled the interactions by simulated dock-ing with HADDOCK (36) and Rosetta FlexPepDock (38) usingthe recently derived FTZ-F1 LBD crystal structure (PDB entry2XHS) and 9-residueMET or GCE peptides generated de novo.Because the interaction between the FTZ-F1 AF2 and the FTZNR box was characterized empirically through the crystalstructure (21), the docking experiments allowed us to comparethe interaction mechanism of the LIXXL motif with that of acanonical LXXLL motif.A comparison of the lowest energy structures of MET and

GCE with the empirically determined structure of FTZ isshown in Fig. 6A. The MET peptide (LYLIENLQK, residues552–560) and GCE peptide (LRLIQNLQK, residues 416–424)are positioned similarly to FTZ peptide along the AF2 grooveformed by helices H3, H3�, H4, and H12 of FTZ-F1. The METand GCE peptide backbones are shifted relative to FTZ (rootmean square deviation of 2.74 and 2.00 Å, respectively), posi-tioning them deeper in the hydrophobic groove and closer toH3 (Fig. 6A). The MET and GCE backbones also show distinctpositioning from one another (root mean square deviation of1.95 Å). The N-terminal half of GCE is shifted closer to theC-terminal end of H12 as compared with MET, whereas theC-terminal half of GCE lies deeper in the hydrophobic grooveand closer to H4 (Fig. 6A).The NR box peptides of MET and GCE are stabilized in the

FTZ-F1 AF2 by contacts with a series of hydrophobic sidechains (Fig. 6, B and C). Residues Leu-554/Leu-558 of MET(Fig. 6B) and Leu-418/Leu-422 of GCE (Fig. 6C), located at the

TABLE 1Secondary structure and conservation of Drosophila MET and GCE

AnnotationCoordinatesa

Structural featuresb Sequence homologycMET GCE

%bHLH 38–90 9–58 NAd 63PAS A 134–185 83–134 �A, �B, �A, �B, �C 40

186–273 135–173 Loop 0Block I 274–283 174–183 �C 70

284–290 184–190 0Block II 291–304 191–203 �D 50

305–364 204–241 Loop 2Block III 365–377 242–254 �E 54PAS B 403–445 269–311 �A�, �B�, �A�, �A�*, �B� 65PAC 446–506 312–371 �C�, �C�, �D�, �E� 66Block IV 507–524 373–390 �IV 61

525–546 391–410 0Block V 547–558 411–422 �V 58C-terminus 559–716 423–689 NCe 0

aFrom D. melanogasterMET and GCE. Coordinates for GCE are given in the original annotation (e.g. see Ref. 56), but note that a new GCE annotation has been proposed(10).

b Secondary structure nomenclature of PAS fold based on Ref. 47.c Percentage of residues whose homology is conserved among proteins from ClustalW multiple sequence alignment: D. melanogaster (MET/GCE), D. grimshawi (MET/GCE), A. aegypti (MET), T. castaneum (MET), and N. vitripennis (MET).

d NA, not analyzed.e NC, no common secondary structures.




�1 and �5 positions of the NR box, are embedded deeplywithin the AF2 groove. Due to the different positioning of thepeptides relative to FTZ-F1, the contacts made by these resi-dues are distinct for MET and GCE. Leu-554 of MET interactswithVal-621, Leu-642, Gln-643, Leu-792, andMet-796 of FTZ-F1, andLeu-558 interactswithMet-639 and the aliphatic regionof Arg-625 (Fig. 6B). Leu-418 of GCE interacts with Val-621,Met-639, Leu-642, Leu-792, and Met-796 of FTZ-F1, whereasLeu-422 interacts with Val-621, Arg-625, Val-635, Gln-638,and Leu-642 (Fig. 6C). A notable feature of bothNRboxes is thepositioning of the isoleucine at the �2 position (Ile-555 inMET, Ile-419 inGCE). For canonical NR boxes, an inner hydro-phobic residue at the�4 position rests along the rim of the AF2groove and interacts with side chains of H3 (20). By contrast,the �2 isoleucine of the MET/GCE NR box rests along theopposing rim and interacts with Met-639 on H4 (Fig. 6, B andC). This mode of interaction is unusual although not novelbecause a similar mechanism was reported for the interactionof ERR� with an LLXXL motif in the coactivator PGC-1� (45).

A characteristic feature of many NR box-AF2 interactions isthe use of a charge clamp, consisting of a conserved glutamicacid residue on H12 and a conserved arginine/lysine on H3;these residues typically form hydrogen bonds with backboneatoms in the core NR box �1 and �5 residues, stabilizing theNR box in the AF2 groove (20). FTZ-F1 utilizes these chargeclamp residues (Arg-625 on H3 and Glu-795 on H12) to formhydrogen bondswith the LXXLLNRbox of FTZ (21). However,we did not observe any charge clamp interactions in the lowestenergy structures for the FTZ-F1�MET and FTZ-F1�GCE inter-actions (Fig. 6,B andC).Moreover, a comprehensive analysis ofinteractions from the top 10 models (Table 2) found no hydro-gen bond formation between FTZ-F1 and the core LIXXL res-idues. The docking experiments therefore predict that the NRbox of MET and GCE requires extensive hydrophobic contactsto interact with FTZ-F1 but does not require the canonicalcharge clamp residues.To test themechanismof interaction identified fromdocking

experiments, point mutations were introduced in FTZ-F1 and

FIGURE 5. Critical MET, GCE residues required for interaction with FTZ-F1. A, alignment of sequences from �IV, �V, and the QR of MET and GCE homologsfrom D. melanogaster, D. grimshawi, A. aegypti, T. castaneum, and N. vitripennis as indicated. Start and end position are shown for each sequence. Identicalresidues are highlighted in black; similar residues are highlighted in gray. Residues targeted for point mutation, including a conserved leucine (Leu-554 in MET,Leu-418 in GCE) in the putative NR box and conserved glutamine/glutamatic acid residues in the QR are indicated with an asterisk. B and C, S2 cells weretransfected with 4� UAS-TATA-Luc reporter construct, along with GAL4 and p65 fusion proteins as indicated (y axis). After transfection, cells were treated withethanol (light gray) or 5.0 � 10�6

M methoprene (dark gray) for 24 h. Luciferase activity was normalized to constitutive �-galactosidase activity (x axis).Interaction with �FTZ-F1 was measured for MET (B) and GCE (C) using the wild type or mutant proteins indicated. Data are shown as the mean � S.D. and arethe result of at least three independent experiments. *, significant difference (p � 0.05). Western blots (IB) at the bottom of each panel show equivalentexpression of wild type and mutant proteins.



were tested in two-hybrid assays. We chose hydrophobic resi-dues from each helix of the AF2 that are predicted to interactwith the core NR boxmotif as well as the charge clamp residuespredicted not to play a role in the interactions with MET andGCE. When individual hydrophobic residues from H3 (Val-621), H4 (Val-635, Met-639, and Leu-642), or H12 (Leu-792andMet-796) of FTZ-F1 aremutated to a charged aspartic acid,the interaction of FTZ-F1 with MET is significantly (p � 0.05)

reduced (Fig. 7A). Mutation of Leu-642 in particular severelyreduced the interaction and was equivalent to the FTZ-F1�H12

deficiency. Although mutation of Leu-792 produced no signifi-cant effect on the interaction of FTZ-F1withGCE,mutating eachof the other hydrophobic residues did significantly reduce thisinteraction (Fig. 7B). The double mutation L792D/M796Dseverely reduced the interactionwithbothMETandGCEandwasalso equivalent to the FTZ-F1�H12 deficiency.Whereas thehydro-

FIGURE 6. Complex formation between the MET, GCE NR box, and the FTZ-F1 LBD. Shown are lowest energy structures from refined docking of MET andGCE NR boxes to the FTZ-F1 LBD (PDB entry 2XHS). AF2 helices from FTZ-F1 (H3, H3�, H4, and H12) are shown in green. NR box peptides for MET (LYLIENLQK,residues 552–560) and GCE (LRLIQNLQK, residues 416 – 424) are shown in orange and red, respectively. In the bottom illustration of each panel, the model isrotated by 60º. A, superposition of MET and GCE peptide backbones with the empirically derived FTZ NR box (STLRALLTN, residues 107–115), shown in blue (20).B and C, molecular details of the FTZ-F1�MET (B) and FTZ-F1�GCE (C) interactions. The core LIXXL residues of MET and GCE and their interacting residues in FTZ-F1are shown. Side chains are colored according to atom type: oxygen (bright red), nitrogen (blue), sulfur (yellow), and carbon (gray for FTZ-F1, orange for MET, redfor GCE).

TABLE 2Summary of MET and GCE residue contacts with FTZ-F1

HADDOCK FlexPepDockHydrogen bonda Hydrophobic contact Hydrogen bond Hydrophobic contact

METLeu-552 Glu-795 Glu-795 Leu-792, Glu-795Tyr-553 Phe-618, Leu-792 Phe-618, Leu-792Leu-554 Met-639, Leu-792, Met-796 Val-621, Leu-642, Gln-643, Leu-792, Met-796Ile-555 Met-639Glu-556Asn-557 Val-621, Arg-625 Val-621, Asp-622Leu-558 Val-635, Gln-638 Arg-625, Met-639Gln-559 Val-635Lys-560 Arg-625 Arg-625

GCELeu-416 Glu-795 Glu-795 Glu-795 Glu-795Arg-417 Asp-614, Glu-795 Phe-618, Thr-791, Leu-792, Glu-795 Gln-790, Thr-791, Leu-792Leu-418 Glu-795 Met-639, Leu-642, Gln-643, Leu-792, Met-796 Val-621, Met-639, Leu-642, Leu-792, Met-796Ile-419 Met-639 Met-639Gln-420Asn-421 Val-621, Arg-625 Phe-618, Arg-625Leu-422 Arg-625, Val-635 Val-621, Arg-625, Val-635, Gln-638, Leu-642Gln-423 Val-635Lys-424 Arg-625 Arg-625

a Listed residue contacts are present in at least 7 of the top 10 docking models.



phobic residues play a similar role in binding both paralogs,muta-tion of residues in H4 or the C-terminal hydrophobic residue inH12 (Met-796) has a greater impact on the interaction with GCEcompared with MET, consistent with the positioning of the GCEpeptide closer to this side of the AF2 groove (Fig. 6A).By sharp contrast, when either of the charge clamp residues

Arg-625 on H3 or Glu-795 on H12 are mutated to alanineFTZ-F1 retains the full interaction with both MET and GCE,and in fact the E795A mutation enhances the interaction withGCE (Fig. 7, A and B). The results from the two-hybrid assaysare thus consistent with the predictions made by dockingexperiments, indicating that FTZ-F1 utilizes hydrophobic res-idues of its AF2 and not the canonical charge clamp to interactwith the novel NR box in MET and GCE.

DISCUSSION

FTZ-F1Mediates JH Signaling throughMechanism Commonto Both Isoforms—FTZ-F1 is a critical transcriptional regulatorin the embryonic, larval, and pupal stages of developmentwhose expression is strictly isoform-specific; �FTZ-F1 is onlypresent in early embryos, whereas �FTZ-F1 is expressed insharp peaks during postembryonic development (40, 41). Wefound that only �FTZ-F1 is present in S2 cells (Fig. 1B), thusimplicating �FTZ-F1 as the isoform responsible for mediatingJH action in this cell line. However, JH induction can be medi-ated by either natural isoform or by FTZ-F1 lacking the iso-form-specific N terminus (Fig. 2), suggesting that an importantcomponent of JH signaling resides in the common domains ofFTZ-F1. Based on our observations that FTZ-F1 is bound in

vivo to multiple enhancers upstream of E75A (12) and interactswith the candidate JH receptors MET and GCE (Fig. 3), wehypothesized that a critical interaction site common to bothisoforms may enable FTZ-F1 to recruit MET or GCE to thetarget promoter. We focused on the FTZ-F1 LBD because thisdomain often plays a critical role in transcriptional regulationby nuclear receptors.We found that disruption of AF2 by trun-cation or point mutations prevents FTZ-F1 from interactingwith either MET or GCE (Figs. 3 and 7). The AF2 is a charac-teristic feature of nuclear receptor LBDs, and its role in recruit-ing transcriptional coregulators is well documented (20). So far,however, the only known function of this domain in FTZ-F1occurs during embryonic development, when it interacts withthe NR box of the homeodomain protein FTZ (21, 30, 31). Wehave now identified an additional function for the FTZ-F1 AF2:mediating JH-dependent transcriptional activation. Becausethe AF2 is present in both isoforms, the findings described heresuggest that JH activation through FTZ-F1 is an isoform-inde-pendent process that could occur during embryogenesisthrough �FTZ-F1 or during postembryonic developmentthrough �FTZ-F1.Candidate JH Receptors MET and GCE Interact JH-depen-

dently with FTZ-F1—Although a clear role for MET in JH reg-ulation is apparent in several insects (3, 5, 6, 8, 12, 15), there isstill significant debate over themolecular basis for JH action. InDrosophila, both MET and GCE mediate sensitivity to exoge-nous JH and mutation of either protein disrupts JH function invivo (11), suggesting that both paralogs may be involved in JH

FIGURE 7. Hydrophobic residues in the FTZ-F1 AF2 are essential to interaction with MET and GCE. S2 cells were transfected with 4� UAS-TATA-Lucreporter and the indicated GAL4 and p65 fusion constructs (y axis). After transfection, cells were treated with ethanol (light gray) or 5.0 � 10�6

M methoprene(dark gray) for 24 h. Luciferase activity was normalized to constitutive �-galactosidase activity (x axis). Interaction of MET (A) and GCE (B) was measured with wildtype and mutant �FTZ-F1 proteins bearing amino acid substitutions (V621D, R625A, V635D, M639D, L642D, L792, M796D, or E795A) or a truncation (�H12) inthe LBD. Data are shown as mean � S.D. from three independent experiments. *, significant (p � 0.05) difference in JH-dependent interaction compared withwild type �FTZ-F1. Western blots below each panel show equivalent expression of wild type and mutant proteins.



signaling. In light of our recent discovery that FTZ-F1 andGCEare required for JH-dependent activation of E75A (12), we wereinterested to determine the role of JH in the interactionbetween FTZ-F1 and the paralogous JH receptor candidates. Byexamining the FTZ-F1�MET and FTZ-F1�GCE interactionsusing a quantitative two-hybrid assay, we found that both para-logs need JH to form heterodimers with FTZ-F1 (Figs. 3, 5, and7). Our results thus show that bothMET and GCE canmediateJH-dependent protein-protein interactions, providing addi-tional evidence that both paralogs are involved in JH signaling.Novel NR Box Enables JH-dependent Interaction of MET and

GCE with FTZ-F1—We previously found that MET and GCEform functional heterodimers with FTZ-F1 that can activatetranscription through the FTZ-F1 response element (12).Because our findings may have important implications inunderstanding the molecular basis for JH signaling, we wereinterested in characterizing the mechanisms driving the FTZ-F1�MET and FTZ-F1�GCE interactions. To identify potentialnuclear receptor interacting motifs, we used homology model-ing and conservation analysis to characterize the secondary andtertiary structure of MET and GCE. One result of this analysiswas the identification of the complete PAS domains for theseproteins. PAS domains are known to function as LBDs withligand typically bound in a pocket formed by the �-sheet and�-helices (14). MET and GCE each possess two tandem PASdomains, andwe found that they both formcanonical folds (Fig.4A). PAS Fold 2 is composed of the previously identified PAS Band adjacent PAC motifs, an arrangement that is typical ofmany PAS domains (18). PAS Fold 1 has a less conventionalstructure because its C-terminal half is made from discreteblocks of conserved sequences interrupted by flexible loops anddoes not form a characteristicmotif. Loops are common in PASdomains; they bind cofactors andmediate intramolecular inter-actions and are thought to provide functional diversity to PAS-containing proteins (18, 51, 52). Interestingly, althoughsequences from structural elements of the PAS folds are con-served (Table 1), the sequence and length of the PAS loops arehighly variable, suggesting that the MET and GCE loops mayconfer functional diversity on homologs in different insects.Overall, characterization of the PAS folds should aid futurestructure-function studies ofMET andGCE in their capacity tobind ligand and form heterodimers.A second result of the in silico studies was the identification

of two �-helices in MET and GCE located C-terminal to PASFold 2. One of these possesses a sequence resembling an NRbox, LIXXL, that is critical to the interactions with FTZ-F1 (Fig.5). There is a growing list of functional NR boxes with non-canonical sequences similar to the LXXLLmotif, having hydro-phobic residues arranged in �1,�2,�5 or �1,�4,�5 orienta-tion and an �-helical structure (43–45). Indeed, dockingsimulations and two-hybrid assays suggest that the conservedLIXXL motif of MET and GCE is a non-conventional NR box(Figs. 6 and 7). Similar to canonical NR boxes like the one foundin FTZ, theMET/GCENR box interacts with hydrophobic res-idues from the AF2; however, it possesses two unusual features.First, the LIXXL motif uses an isoleucine residue at the �2position to interact with residues on H4 of the AF2 (Fig. 6); bycontrast, LXXLL motifs use a �4 leucine to interact with resi-

dues on H3 (20). Second, the charged residues Glu-795 andArg-625 of FTZ-F1 that form hydrogen bonds with the peptidebackbone of canonical NR boxes, such as the one in FTZ (21),are dispensable to the interactions with MET and GCE (Figs. 6and 7), suggesting these interactions are formed primarilythroughhydrophobic residues. These two findings indicate thatMET and GCE utilize a non-canonical NR box whose mecha-nistic basis of interaction differs from the traditional LXXLL-type NR box. Our finding is consistent with emerging evidencethat diverse modes of interaction exist for NR box-AF2 com-plexes. An interesting example of this comes from the coacti-vator PGC1-�, which possesses two functional NR boxes. Acanonical LXXLL NR box interacts with TR�1 and PPAR�through a mechanism that does not require charged residues(53, 54); a second, non-canonical LLXXLNR box interacts withERR� through a charge clamp-dependent mechanism (45). Toour knowledge, theMET/GCEmotif represents the first exam-ple of an NR box that is both non-canonical in sequence andutilizes a charge clamp-independent mode of interaction.Functional Divergence Is Evident in C Terminus of MET and

GCE—Gene duplication is an important mechanism allowinggene products to assume new functional roles over time (55).The Met gene likely arose by duplication of ancestral gce priorto the origination of the Drosophila genus (56), and consistentwith an evolutionarily recent duplication, MET and GCE sharesubstantial sequence similarity in their bHLH and PASdomains and are largely redundant in vivo (8–10). In our study,we found that in addition to the NR box, there is a QR region inthe C terminus that has paralog-specific function and is usedonly by MET as a secondary nuclear receptor interaction site(Fig. 5). Inspection of the QR region in drosophilids reveals thatin addition to distinct positioning of the QR within the proteins(Fig. 4B) there are clear differences in structure and conserva-tion; the QR of MET is significantly shorter (�30 residues) andcontains polyglutamine tracts similar to those found in someSRC family members (26–29), whereas the QR of GCE is over70 residues in length and is homologous to aQR region found inmosquito MET (supplemental Fig. S3). In addition, the METand GCE possess paralog-specific acidic and P/S regions in theC terminus (Fig. 4B and supplemental Fig. S3). Because the invivo functions of MET and GCE are not entirely redundant (9),we suggest that functional divergence may be attributed in partto the paralog-specificQR regions and other domains located inthe C termini of MET and GCE.In summary, we have examined themolecular basis of JH-de-

pendent interaction between the nuclear receptor FTZ-F1 andthe bHLH-PAS proteins MET and GCE. A novel NR boxenables both MET and GCE to interact JH-dependently withthe AF2 of FTZ-F1. We suggest that this mechanism functionsin the JH transcription activation of E75A and may play a sig-nificant role in mediating JH signaling in vivo.

Acknowledgments—We appreciate the gift of fusion protein expres-sion vectors and luciferase reporter vector from Dr. Takahiro Kusak-abe (Kyushu University, Japan). We also acknowledge VeronicaDubrovskaya for providing technical assistance and for critical read-ing of the manuscript.





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