Interaction patterns of methoprene-tolerant and germ cell-expressed Drosophila JH receptors suggest significant differences in their functioning

Methoprene-tolerant (Met) and germ cell-expressed (Gce) proteins were shown to be juvenile hormone (JH) receptors of Drosophila melanogaster with partially redundant functions. We raised the question of where the functional differentiation of paralogs comes from. Therefore, we tested Met and Gce interaction patterns with selected partners. In this study, we showed the ability of Gce and its C-terminus (GceC) to interact with 14-3-3 in the absence of JH. In contrast, Met or Met C-terminus (MetC) interactions with 14-3-3 were not observed. We also performed a detailed structural analysis of Met/Gce interactions with the nuclear receptor fushi tarazu factor-1 (Ftz-F1) ligand-binding domain. We showed that GceC comprising an Ftz-F1-binding site and full-length protein interacts with Ftz-F1. In contrast to Gce, only MetC (not full-length Met) can interact with Ftz-F1 in the absence of JH. We propose that the described differences result from the distinct tertiary structure and accessibility of binding sites in the full-length Met/Gce. Moreover, we hypothesize that each interacting partner can force disordered MetC and GceC to change the structure in a partner-specific manner. The observed interactions seem to determine the subcellular localization of Met/Gce by forcing their translocation between the nucleus and the cytoplasm, which may affect the activity of the proteins. The presented differences between Met and Gce can be crucial for their functional differentiation during D. melanogaster development and indicate Gce as a more universal and more active paralog. It is consistent with the theory indicating gce as an ancestor gene

The intrinsically disordered region of GCE protein adopts a more fixed structure by interacting with the LBD of the nuclear receptor FTZ-F1.

The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family. [MediaObject not available: see fulltext.

Intrinsic Disorder of the C-Terminal Domain of <i>Drosophila</i> Methoprene-Tolerant Protein

<div><p>Methoprene tolerant protein (Met) has recently been confirmed as the long-sought juvenile hormone (JH) receptor. This protein plays a significant role in the cross-talk of the 20-hydroxyecdysone (20E) and JH signalling pathways, which are important for control of insect development and maturation. Met belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family of transcription factors. In these proteins, bHLH domains are typically responsible for DNA binding and dimerization, whereas the PAS domains are crucial for the choice of dimerization partner and the specificity of target gene activation. The C-terminal region is usually responsible for the regulation of protein complex activity. The sequence of the Met C-terminal region (MetC) is not homologous to any sequence deposited in the Protein Data Bank (PDB) and has not been structurally characterized to date. In this study, we show that the MetC exhibits properties typical for an intrinsically disordered protein (IDP). The final averaged structure obtained with small angle X-ray scattering (SAXS) experiments indicates that intrinsically disordered MetC exists in an extended conformation. This extended shape and the long unfolded regions characterise proteins with high flexibility and dynamics. Therefore, we suggest that the multiplicity of conformations adopted by the disordered MetC is crucial for its activity as a biological switch modulating the cross-talk of different signalling pathways in insects.</p></div