The molecular basis of conformational instability of the ecdysone receptor DNA binding domain studied by in silico and in vitro experiments.
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.
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Agnieszka
A
Szamborska-Gbur
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Grzegorz
G
Rymarczyk
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Marek
M
Orłowski
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Tomasz
T
Kuzynowski
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Michał
M
Jakób
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Agnieszka
A
Dziedzic-Letka
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.
Andrzej
A
Górecki
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Piotr
P
Dobryszycki
Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.
Andrzej
A
Ożyhar
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D029721:Drosophila Proteins / Q000737:chemistry*; D004331:Drosophila melanogaster; D057927:Hydrophobic and Hydrophilic Interactions; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry*; D044366:Transition Temperature
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; D004247:DNA