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

Abstract

The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein–DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5′- and/or 3′-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5′-end of the regulatory element and moved close to the 3′-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented.

Keywords:

• nuclear receptor
• Usp
• EcR
• DHR38
• intein
• FRET
• conformational change
• DNA bending

Acknowledgments

The technical assistance of Mrs. M. Ostrowska is gratefully acknowledged. This work was financed by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wrocław University of Technology.

Notes

aSubscript 5 means that the fluorescent dye was at the hsp27pal 5′-end, whereas 3 means that the dye was at the 3′-end.

bΔL means that hsp27pal was mutated at the 5′-half-site, whereas ΔR means that hsp27pal was mutated at the 5′-half-site.

cData calculated from the fluorescence spectra obtained for singly labeled DNA with TMRh probe of final concentration 40 nM titrated with a 35 μM protein stock solution up to a final EcRDBDf or EcRDBDf/UspDBD concentration of about 60 nM and incubated for 10 min for each titration step. Each time a control titration of the unlabeled DNA with the FL-labeled protein was done.

aData calculated from the fluorescence spectra obtained for singly labeled DNA with TMRh probe of final concentration 40 nM titrated with a 35 μM protein stock solution up to a final UspDBDf or UspDBDf/UspDBD concentration of about 60 nM and incubated for 10 min for each titration step. Each time a control titration of the unlabeled DNA with the FL-labeled protein was done.

aEach time a control titration of the unlabeled DNA with the FL-labeled protein was done. Equilibrium titration of doubly labeled hsp27 (of final concentration 40 nM) with equimolar concentration of EcRDBD or EcRDBD-DHR38 mixture in the range of concentration 0–450 nM and incubated for 10 min for each titration step. The donor–acceptor distance was determined after full saturation was reached.

bData taken from Dobryszycki et al. (2006).