ABSTRACT
The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule.
Subject(s)
DNA-Binding Proteins/chemistry , Drosophila Proteins/chemistry , Drosophila melanogaster , Molecular Dynamics Simulation , Receptors, Steroid/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites , DNA/chemistry , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Unfolding , Receptors, Steroid/genetics , Transition TemperatureABSTRACT
Intrinsically disordered proteins (IDPs) belong to the newly discovered and still growing group of proteins. In contrast to globular proteins IDPs fail to fold into a well-defined tertiary structure under physiological conditions and they are characterized by extraordinary structural flexibility and plasticity. These features enable IDPs to adopt different conformations in response to different stimuli or different partners. Additionally, a pliable polypeptide chain, much more accessible in IDPs, causes that IDPs can undergo extensive posttranslational modifications which might lead to further modulation of IDPs conformation enabling rapid regulation of IDPs activity. In this way IDPs are involved in regulation of various regulatory pathways and promoting the assembly of supramolecular complexes. IDPs discovery reveals a new face of proteins and constitutes a new challenge for modern biochemistry.
Subject(s)
Proteins/chemistry , Proteins/metabolism , Humans , Molecular Conformation , Protein Conformation , Protein FoldingABSTRACT
Drosophila hormone receptor 38 (dHR38), an ortholog of the vertebrate NR4A subclass of nuclear receptors, responds to ecdysteroids, which mediate developmental transitions during the Drosophila life cycle. However, this response is independent of the ecdysteroid receptor, and it does not involve binding of ecdysteroids to dHR38. It has been suggested that ecdysteroids may indirectly activate dHR38, perhaps by recruiting specific proteins. There have been recent reports pointing out the decisive role that nuclear receptor N-terminal domains (NTDs) have in protein-protein interactions that are important for regulation of gene expression. It is reasonable to assume that dHR38-NTD may also be involved in some protein-protein interactions that are critical for the ecdysteroid signaling pathway. To facilitate the exploration of the molecular basis of these interactions, we developed and optimized a protocol for the efficient expression and purification of the recombinant dHR38-NTD. Using a diverse array of biochemical and biophysical methods, we carried out the first structural characterization of dHR38-NTD. The results of our study indicate that dHR38-NTD exhibits a characteristic reminiscent of pre-molten globule-like intrinsically disordered proteins existing in a partially unfolded conformation with regions of secondary structures. The dHR38-NTD structure, which apparently comprises some local, ordered, tertiary structure clusters, is pliable and can adopt more ordered conformations in response to changes in environmental conditions. Thus, dHR38-NTD, which exhibits the structural and functional characteristic of a pre-molten globule-like intrinsically disordered protein, could serve as a platform for multiple protein-protein interactions, possibly including interactions with proteins involved in an unusual ecdysteroid signaling pathway.
