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

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 A distance obtained for the UspDBD-complexed DNA a bend of about 23.1 +/- 2.9 degrees was measured. Nearly the same value (23.0 +/- 3.4 degrees) 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 degrees and 20.9 +/- 3.6 degrees . 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.

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element

“…In particular, it has been shown that when UspDBD preferentially binds the 5′-half-site of hsp27 pal , it acts as an anchor dictating the polarity of the heterocomplex (5′-UspDBD-EcRDBD-3′) (Jakób et al, 2007;Niedziela-Majka, Kochman, & Ożyhar, 2000;Orłowski et al, 2004;Ozyhar et al, 1991). We had also shown in our previous work that hsp27 pal bound by the UspDBD/EcRDBD heterocomplex is deformed (Dobryszycki et al, 2006;Jakób et al, 2007). Moreover, both proteins interacted with this regulatory element in a cooperative manner (Krusiński, Oży-har, & Dobryszycki, 2010).…”

“…The double-stranded DNA was annealed by hybridizing equal amounts of the labeled single-stranded oligonucleotides (Dobryszycki et al, 2006). The double-stranded DNA was labeled with FL (donor) at the 5′-end and TMRh fluorophore (acceptor) at the 3′-end.…”

“…Fluorescence spectra were recorded with a All measurements were carried out at 25°C. DNA bend angles were estimated based on the FRET steadystate measurements as described previously (Dobryszycki et al, 2006). Fluorescence lifetimes were determined with a multifrequency phase fluorometer, SLM 48000S (SLM Aminco, Urbana, IL).…”

“…Moreover, both proteins interacted with this regulatory element in a cooperative manner (Krusiński, Oży-har, & Dobryszycki, 2010). The use of three independent methods, fluorescence resonance energy transfer (FRET), electrophoretic mobility shift assay (EMSA), and X-ray crystallography, enabled us to demonstrate that the interaction between the UspDBD and the hsp27 pal sequence induced bending of about 23° (Dobryszycki et al, 2006). Interestingly, subsequent binding of the EcRDBD molecule, i.e.…”

“…In order to accomplish this, FL (donor) was attached to the EcRDBD or UspDBD C-terminal residues and TMRh (acceptor) was attached to the ends of hsp27. Previous studies indicated that there were differences in the molecular mechanisms used to recognize hsp27 pal by isolated UspDBD or UspDBD together with EcRDBD (Dobryszycki et al, 2006;Niedziela-Majka, Kochman, & Ożyhar, 2000;Orłowski et al, 2004). It was demonstrated that UspDBD binds preferentially the 5′-half-site of hsp27 pal , whereas the UspDBD/ EcRDBD complex is formed in a synergistic manner on the regulatory element.…”