Articles The DNA-Binding Domain of the Ultraspiracle Drives Deformation of the Response Element Whereas the DNA-Binding Domain of the Ecdysone Receptor Is Responsible for a Slight Additional Change of the Preformed Structure ² Piotr Dobryszycki, ‡ Iwona Grad, ‡,§ Tomasz Krusin ´ski, Piotr Michaluk, | Dorota Sawicka, Agnieszka Kowalska, Marek Orlowski, Michal Jako ´b, Grzegorz Rymarczyk, Marian Kochman, and Andrzej Oz ˘ yhar* DiVision of Biochemistry, Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw UniVersity of Technology, Wybrzez ˘ e Wyspian ´ skiego 27, 50-370 Wroclaw, Poland ReceiVed July 13, 2005; ReVised Manuscript ReceiVed October 28, 2005 ABSTRACT: Ecdysteroids control molting and metamorphosis in insects via a heterodimeric complex of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle protein (Usp). We used fluorescence resonance energy transfer (FRET) to study the topology of the natural pseudopalindromic element from the hsp27 gene (hsp27pal) in complex with the DNA-binding domains of Usp and EcR (UspDBD and EcRDBD, respectively). Steady-state data revealed shortening of the end-to-end distance of the hsp27pal- derived probe. For the 70.8 ( 0.6 Å distance obtained for the UspDBD-complexed DNA a bend of about 23.1 ( 2.9° was measured. Nearly the same value (23.0 ( 3.4°) was obtained for the DNA complexed with the UspDBD/EcRDBD heterodimer. The respective bend angles estimated using fluorescence decay measurements were 19.0 ( 2.1° and 20.9 ( 3.6°. Thus, the FRET data suggest for the first time that the UspDBD defines the architecture of the UspDBD/EcRDBD heterocomplex due to the significant deformation of the hsp27pal. This suggestion has been further reinforced using gel retardation experiments, which, in conjunction with high-resolution DNase I footprinting, indicate that the main contribution to the observed bend is given by the UspDBD itself, while binding of the EcRDBD molecule brings on a slight additional change of the preformed structure. Nuclear receptors constitute a large group of transcription factors that are involved in many important biological processes. The receptors exert their action via binding to specific DNA sequences called response elements either as monomers or as dimers (1). For some members of this largest superfamily of metazoan transcription factors it has been shown that they induce a substantial distortion in the DNA structure, which may influence the transcription-inducing activity of the complex (2-6). Ecdysteroids are hormones that function as the major inducing signals responsible for regulation of postembryonic development in insects and possibly in other arthropods (7). The functional receptor for ecdysteroids is a transcription factor comprised of two nuclear receptors, the ecdysone receptor (EcR, 1 NR1H1) and a homologue of the mammalian retinoid X receptor, the ultraspiracle protein (Usp, NR2B4) (8, 9). The Usp/EcR heterodimer binds ecdysone response elements after binding to the steroid hormone 20-hydroxyecdysone (20E) and consequently stimulates transcription of targeted genes (10). It has also been shown that the Usp/EcR heterodimer inhibits transcription in its unliganded state (11). Although molecular studies of the Usp/EcR heterodimer are much less extensive than those of vertebrate heterodimeric receptors, it is already clear that the ecdysteroid receptor complex, which exhibits mixed-type characteristics typical for both steroid and nonsteroid receptors, holds an exceptional position among the nuclear receptor family. One of its most intriguing features is propensity for response elements arranged as highly degenerated palindromes with a single intervening nucleotide [see Niedziela-Majka et al. (12) for a review]. This clearly distinguishes the Usp/EcR complex from vertebrate counterparts which tend to form complexes on inherently asymmetric DNA-binding sites composed of directly repeated half-sites (13). Our mutational studies on the interaction of the Usp and the EcR DBDs (UspDBD and EcRDBD, respectively) with the highly degenerated pseudopal- ² This research has been funded by a grant (3P04B 009 23) from the Polish State Committee for Scientific Research. * Corresponding author. Phone: +48 (71) 3206333. Fax: +48 (71) 3206337. E-mail: andrzej.ozyhar@pwr.wroc.pl. ‡ The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint first authors. § Present address: De ´partement de Biologie Cellulaire, Universite ´ de Gene `ve, 30, Quai Ernest-Ansermet, CH-1211 Gene `ve 4, Switzerland. | Present address: The Nencki Institute of Experimental Biology, Pasteura 3, 02-093 Warsaw, Poland. 1 Abbreviations: Cy-5, carboxymethylindocyanine N-hydroxysuc- cinimidyl ester; DBD, DNA-binding domain; EcR, ecdysone receptor; EcRDBD, EcR DNA-binding domain; 20E, 20-hydroxyecdysone; FRET, fluorescence resonance energy transfer; FL, fluorescein; hsp27, DNA sequence encoding hsp27 protein; hsp27pal, natural 20-hydroxy- ecdysone response element consisting of an imperfect palindrome from the promoter region of the Drosophila hsp27 gene; IR-1, idealized element organized as an inverted repeat separated by 1 bp; TMRh, tetramethylrhodamine; Usp, ultraspiracle protein; UspDBD, Usp DNA- binding domain. 668 Biochemistry 2006, 45, 668-675 10.1021/bi051354b CCC: $33.50 © 2006 American Chemical Society Published on Web 12/22/2005