Two proteins, the ecdysone receptor (EcR) and a homologue of the mammalian retinoid X receptor, the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Both proteins are members of the nuclear receptor superfamily which are ligand-inducible transcription factors. In the presence of 20-hydroxyecdysone (20E) - the steroid hormone, the Usp/EcR heterodimer is formed via DNA-binding domains (UspDBD and EcRDBD, respectively) on the natural pseudopalindromic element from the hsp27 gene promoter. The UspDBD defines the architecture of the UspDBD-EcRDBD heterocomplex due to the binding to the 5’-halfsite of the hsp27 and the deformation of the response element. The main contribution to the bend is given by the UspDBD, while the EcRDBD molecule brings on a slight conformational change (Dobryszycki et al., Biochemistry 2006, vol. 45).

In this communication we examined the effect of the EcRDBD on the formation of the EcRDBD-UspDBD-hsp27 complex in solution using FRET measurements. We used fluorescence resonance energy transfer (FRET) to study the topology of the DNA-binding domains of Usp and EcR in complex with the hsp27. For the measurements of the distances between fluorescence donor and acceptor pairs UspDBD was labeled using an intein method with the fluorescent Cys-Lys-fluorescein dipeptide on the C-terminal end (donor label). 5’ or 3’-ends of the hsp27 element were labeled with TAMRA fluorofore (acceptor label). Then, the distance between the UspDBD C-terminus and both hsp27 ends were determined. In the absence and in the presence of EcRDBD the distances for the 5’-end were 65.5 +/- 3.4 Å and 71.8 +/- 2.0 Å, respectively. The distances between the 3’-end and UspDBD C-terminus were 66.1 +/- 1.3 Å and 72.8 +/- 1.6 Å, respectively. Based upon above presented results we conclude that binding of the EcRDBD induces strong conformational change of UspDBD, whereas UspDBD causes only deformation of the response element. We compared the “FRET” results with the recently obtained, in our laboratory, data from X-ray experiments (Jakób et al., Nucleic Acid Res. 2007).

This work was supported by the grant from Polish Ministry of Science and Higher Education 3552/P01/2006/31.

• Legal notice:
  

  Copyright (c) Pielaszek Research, all rights reserved.
  The above materials, including auxiliary resources, are subject to Publisher's copyright and the Author(s) intellectual rights. Without limiting Author(s) rights under respective Copyright Transfer Agreement, no part of the above documents may be reproduced without the express written permission of Pielaszek Research, the Publisher. Express permission from the Author(s) is required to use the above materials for academic purposes, such as lectures or scientific presentations.
  In every case, proper references including Author(s) name(s) and URL of this webpage: https://science24.com/paper/10849 must be provided.