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

Abstract

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.

Juvenile Hormone Membrane Signaling Enhances its Intracellular Signaling Through Phosphorylation of Met and Hsp83

“…With regard to the regulation of Met subcellular localization, the JH-dependent nuclear localization signal in the PAS-B+ domain may outweigh phosphorylation modification ( Greb-Markiewicz et al, 2011 ). Previous studies have shown that the JH-dependent nuclear import of the PAS-B+ domain of Met is responsible for binding of the nuclear receptor Ftz-F1 ( Bernardo and Dubrovsky, 2012 ; Kolonko et al, 2020 ). Conceivably, loss of phosphorylation modification in the PAS-B+ domain of Met could have no effect on JH intracellular signaling activity in Drosophila , differing from the report that T393 phosphorylation in the PAS-B domain is critical for Kr-h1 transcription in H. armigera ( Li et al, 2021 ).…”

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation

“…The C-terminal NLSs reside in proline/serine/threonine-rich regions that are intrinsically disordered in both D. melanogaster GCE ( 69 , 72 ) and T. castaneum methoprene-tolerant proteins ( Fig. 7 ).…”

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

“…All experiments were performed as described previously ( Kolonko et al, 2020 ). In short, African green monkey kidney fibroblast COS-7 cells (ATCC CRL-1651) were grown in Ø6 cm plates at 37°C 24 h prior to transfection (Xfect Transfection Reagent Takara Bio) with 9 μg of the appropriate vector encoding the selected protein, or with 6 μg of each from two selected vectors in the case of co-transfection.…”

“…In contrast to bHLH and PAS domains presenting high sequence homology (78% for bHLH, 68% for PAS-1, and 86% for PAS-2) ( Moore et al, 2000 ), the C-termini of Met and Gce (MetC and GceC, respectively) are highly differentiated ( Supplementary Figure S1 , based on the SIM server ( Huang and Miller, 1991 )) ( Moore et al, 2000 ; Kolonko et al, 2016 ; Kolonko et al, 2020 ). Variable C-terminal fragments of bHLH-PAS proteins were shown to comprise transactivation domains (TADs, as described by Moffett and Pelletier (2000 )) and are considered important elements modulating protein activity ( Kewley et al, 2004 ; Furness et al, 2007 ).…”