Crystal Structure of Fushi Tarazu Factor 1 Ligand Binding Domain/Fushi Tarazu Peptide Complex Identifies New Class of Nuclear Receptors

Abstract

The interaction between the orphan nuclear receptor FTZ-F1 (Fushi tarazu factor 1) and the segmentation gene protein FTZ is critical for specifying alternate parasegments in the Drosophila embryo. Here, we have determined the structure of the FTZ-F1 ligand-binding domain (LBD)⅐FTZ peptide complex using x-ray crystallography. Strikingly, the ligand-binding pocket of the FTZ-F1 LBD is completely occupied by helix 6 (H6) of the receptor, whereas the cofactor FTZ binds the co-activator cleft site of the FTZ-F1 LBD. Our findings suggest that H6 is essential for transcriptional activity of FTZ-F1; this is further supported by data from mutagenesis and activity assays. These data suggest that FTZ-F1 might belong to a novel class of ligand-independent nuclear receptors. Our findings are intriguing given that the highly homologous human steroidogenic factor-1 and liver receptor homolog-1 LBDs exhibit sizable ligand-binding pockets occupied by putative ligand molecules.FTZ-F1 (Fushi tarazu factor 1)is a member of the orphan nuclear receptor (NR) 3 family, NR5A3, which interacts with the homeodomain protein FTZ to define alternate parasegmental regions in the Drosophila embryo (1-4). FTZ-F1 was originally isolated as a putative transcriptional activator of the ftz (fushi tarazu) gene (5). In blastoderm stage embryos, FTZ-F1 is uniformly distributed, whereas FTZ is expressed in seven stripes representing even-numbered parasegments (6). Both proteins are required for the formation of these even-numbered parasegments (7). FTZ-F1 has two conserved structural domains: a DNA-binding domain consisting of two zinc fingers and a putative ligandbinding domain (LBD) at the C terminus. Sequence alignments with other NRs and secondary structure predictions suggest that the FTZ-F1 LBD, like that of other NR proteins, is composed of 12 helices. Recent reports have also shown that transcriptional regulation by ligand-dependent NRs is achieved by interactions with cognate lipophilic ligands, which convert apo-NRs into the holo-conformation (8 -14) and lead to transcriptional activation (15, 16) or repression (17,18). These studies suggest that ligand binding to NR LBDs induces a conformational change in the LBD such that the AF-2 helix, which is the last ␣-helix at the C terminus, reorients its position to facilitate the binding of cofactors. However, FTZ-F1 has been considered an orphan NR because no cognate ligand has yet been identified. Therefore, the molecular mechanism governing FTZ-F1 activation and regulation has remained largely unknown.Among the vertebrate NRs, those most similar to FTZ-F1 are steroidogenic factor-1 (SF-1/NR5A1) and liver receptor homologue-1 (LRH-1/NR5A2), which share ϳ40% sequence identity with FTZ-F1 in the LBD. Recently, the structure of the mouse LRH-1 LBD has been solved, revealing that the AF-2 helix forms an active conformation despite the presence of a large and empty ligand-binding pocket (LBP) (19). Unlike mouse LRH-1, human LRH-1 requires the putative ligand phosphatidylinositol for optimum activity (...

Divergent Sequence Tunes Ligand Sensitivity in Phospholipid-regulated Hormone Receptors

“…Finally, we show by mass spectrometry and lipid phosphorous assays that dmFtz-F1 is devoid of PLs when expressed and purified from E. coli, supporting the claim that that dmFtz-F1 is a truly constitutive receptor (41). The selective advantage for PL-independent Ftz-F1 signaling in arthropods is unclear.…”

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

“…The N-terminal bHLH and PAS domains of SRC proteins are dispensable to their interaction with nuclear receptors (23)(24)(25), whereas a Q R region in the C terminus is used as a second interaction site through a poorly understood mechanism (26 -29). In addition to the SRC family, NR boxes have been identified in a variety of other coregulators, including the homeodomain protein FTZ, which utilizes an NR box to interact with the AF2 of FTZ-F1 (21,30,31). No functional NR boxes have yet been identified in MET or GCE, so the mechanism of their interaction with FTZ-F1 remains unresolved.…”

“…1A). The LBD of FTZ-F1 has a typical structure consisting of 12 ␣-helices and an anti-parallel ␤-sheet; it forms a coactivator interaction surface referred to as activation function 2 (AF2) from helices H3, H3Ј, H4, and H12 (20,21). In contrast to ligand-dependent AF2, the AF2 of FTZ-F1 is stabilized in an active conformation by H6, which serves as a pseudoligand and may allow FTZ-F1 to function as a ligand-independent activator (21).…”

“…The LBD of FTZ-F1 has a typical structure consisting of 12 ␣-helices and an anti-parallel ␤-sheet; it forms a coactivator interaction surface referred to as activation function 2 (AF2) from helices H3, H3Ј, H4, and H12 (20,21). In contrast to ligand-dependent AF2, the AF2 of FTZ-F1 is stabilized in an active conformation by H6, which serves as a pseudoligand and may allow FTZ-F1 to function as a ligand-independent activator (21). The presence of a constitutively active AF2 suggests that FTZ-F1 could act primarily through protein-protein interactions, and our finding that FTZ-F1 interacts with both MET and GCE (12) raises the possibility that FTZ-F1 mediates JH action indirectly through the formation of FTZ-F1⅐MET or FTZ-F1⅐GCE heterodimers.…”

Solution Structure of LXXLL-related Cofactor Peptide of Orphan Nuclear Receptor FTZ-F1

“…23 Thus, it is assumed that FTZ uses the same LXXLL-related motifs to interact with FTZ-F1. Recently, our laboratory reported the structural basis for the FTZ-F1/FTZ interaction by X-ray crystallography, 3 however, the cofactor peptide shows a short helical structure. To gain further insight into the structural properties of the cofactor peptide in solution state, we have performed NMR studies on a synthetic peptide containing LXXLL-related motifs of FTZ, which is corresponding to FTZΔ112-120 (FTZ PEP ).…”