title: The Drosophila juvenile hormone receptor candidates methoprene-tolerant (MET) and germ cell-expressed (GCE) utilize a conserved LIXXL motif to bind the FTZ-F1 nuclear receptor.
abstract: Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains controversial. We previously found that in Drosophila Schneider 2 cells, the nuclear receptor FTZ-F1 is required for activation of the E75A gene by JH. Here, we utilized insect two-hybrid assays to show that FTZ-F1 interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE, in a JH-dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 activation function 2 (AF2) is removed, implicating AF2 as an interacting site. Through homology modeling, we found that MET and GCE possess a C-terminal α-helix featuring a conserved motif LIXXL that represents a novel nuclear receptor (NR) box. Docking simulations supported by two-hybrid experiments revealed that FTZ-F1·MET and FTZ-F1·GCE heterodimer formation involves a typical NR box-AF2 interaction but does not require the canonical charge clamp residues of FTZ-F1 and relies primarily on hydrophobic contacts, including a unique interaction with helix 4. Moreover, we identified paralog-specific features, including a secondary interaction site found only in MET. Our findings suggest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1.
authors.affiliation: Department of Biology, Fordham University, Bronx, New York 10458 , USA.
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Bernardo
authors.forename: Edward B
authors.initials: EB
authors.lastname: Dubrovsky
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D007605:Juvenile Hormones / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D007605:Juvenile Hormones; D014157:Transcription Factors; C111234:MET protein, Drosophila; C543965:gce protein, Drosophila; C499712:nuclear hormone receptor FTZ-F1, Drosophila