Title |
The Molecular Basis of Conformational Instability of the Ecdysone Receptor DNA Binding Domain Studied by In Silico and In Vitro Experiments
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Published in |
PLOS ONE, January 2014
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DOI | 10.1371/journal.pone.0086052 |
Pubmed ID | |
Authors |
Agnieszka Szamborska-Gbur, Grzegorz Rymarczyk, Marek Orłowski, Tomasz Kuzynowski, Michał Jakób, Agnieszka Dziedzic-Letka, Andrzej Górecki, Piotr Dobryszycki, Andrzej Ożyhar |
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. |
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