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Molecular Endocrinology, doi:10.1210/me.2004-0154
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Molecular Endocrinology 18 (9): 2166-2184
Copyright © 2004 by The Endocrine Society

Plasticity of the Ecdysone Receptor DNA Binding Domain

Marek Orlowski, Monika Szyszka, Agnieszka Kowalska, Iwona Grad, Anna Zoglowek, Grzegorz Rymarczyk, Piotr Dobryszycki, Daniel Krowarsch, Fraydoon Rastinejad, Marian Kochman and Andrzej Ozyhar

Institute of Organic Chemistry, Biochemistry and Biotechnology (M.O., M.S., A.K., I.G., A.Z., G.R., P.D., M.K., A.O.), Division of Biochemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland; Laboratory of Protein Engineering (D.K.), Institute of Biochemistry and Molecular Biology, University of Wroclaw, 50-137 Wroclaw, Poland; and Department of Pharmacology (F.R.), Department of Molecular Genetics and Biochemistry, University of Virginia Health System, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Andrzej Ozyhar, Institute of Organic Chemistry, Biochemistry and Biotechnology, Division of Biochemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50–370 Wroclaw, Poland. E-mail: andrzej.ozyhar{at}pwr.wroc.pl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ecdysteroids coordinate molting and metamorphosis in insects via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and the ultraspiracle (Usp) protein. Here we show how the DNA-recognition {alpha}-helix and the T box region of the EcR DNA-binding domain (EcRDBD) contribute to the specific interaction with the natural response element and to the stabilization of the EcRDBD molecule. The data indicate a remarkable mutational tolerance with respect to the DNAbinding function of the EcRDBD. This is particularly manifested in the heterocomplexes formed between the EcRDBD mutants and the wild-type Usp DNA-binding domain (UspDBD). Circular dichroism (CD) spectra and protein unfolding experiments indicate that, in contrast to the UspDBD, the EcRDBD is characterized by a lower {alpha}-helix content and a lower stability. As such, the EcRDBD appears to be an intrinsically unstructured protein-like molecule with a high degree of intramolecular plasticity. Because recently published crystal structures indicate that the ligand binding domain of the EcR is also characterized by the extreme adaptability, we suggest that plasticity of the EcR domains may be a key factor that allows a single EcR molecule to mediate diverse biological effects.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE 20-HYDROXYECDYSONE (20E) is a steroid hormone that regulates molting, metamorphosis, reproduction, and many other developmental processes in insects and in other arthropods (1). In insects 20E functions through a heterodimeric receptor complex comprised of the ecdysone receptor (EcR) and the ultraspiracle (Usp) (2, 3). Both proteins are members of the nuclear receptor superfamily that includes receptors for steroid and thyroid hormones, and retinoids and vitamin D, as well as many other ligand-inducible transcription factors. Nuclear receptors act by interacting with DNA response elements of target genes known as hormone response elements. The most known natural 20E-response elements are highly degenerated imperfect palindromes with a single intervening nucleotide (4, 5, 6, 7, 8, 9). The best characterized is the pseudopalindromic element from the heat shock protein 27 gene promoter (hsp27pal), which is composed of two heptameric half-site sequences separated by one central base pair (4, 10). Analysis of the interaction of the Usp and the EcR DNA-binding domains (DBDs) (UspDBD and EcRDBD, respectively) indicated that the UspDBD acts as a specific anchor that preferentially binds the 5' half-site, and thus is a key factor dictating the polarity (i.e. 5'-UspDBD-EcRDBD-3') of the heterocomplex on the hsp27pal element (11). Interestingly, this arrangement was recently observed in the crystal structure of the UspDBD/EcRDBD complex bound to an idealized element organized as an inverted repeat of the 5'-AGGTCA-3' sequence separated by 1 bp (IR-1) (12). The functional significance of the UspDBD -hsp27pal interaction has been supported by the mutational experiments conducted in our laboratory. These studies demonstrated that changes in the UspDBD binding affinity, caused by the substitution of the amino acids in the DNA-recognition {alpha}-helix by alanine, are directly reflected in the UspDBD/EcRDBD-hsp27pal interaction pattern (13).

In this report we illustrate how the DNA-recognition {alpha}-helix of the EcRDBD contributes to the specific interaction with the hsp27pal and to the stabilization of the EcRDBD protein fold. In contrast to the UspDBD, the EcRDBD exhibits unexpected mutational tolerance in its DNA-recognition {alpha}-helix, with many mutations still allowing efficient formation of the complex on the response element. The mutational tolerance is further seen for the T box region of the EcRDBD, which readily accepts substitution of almost all amino acids by alanine, when the EcRDBD is complexed with the UspDBD on the hsp27pal. These suggest that the EcRDBD behaves in many ways as a protein characterized by high intramolecular flexibility/plasticity. Interestingly, recently published crystallographic data indicate that the ligand binding domain of the EcR is also characterized by a high degree of ligand tolerance and adaptability, a property that allows targeting by ligands belonging to distinct chemical classes, including both steroid and nonsteroidal agonists (14). Thus, the EcR seems to be the first example of the nuclear receptor having two flexible domains. The possible biological significance of this structural plasticity is discussed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Amino Acid Substitutions in the DNA-Recognition {alpha}-Helix of the EcRDBD on the Interaction with the hsp27pal Element
To identify the critical amino acids involved in specific binding of the hsp27 response element by the EcRDBD, we have substituted each of nine selected amino acids of the putative DNA-recognition {alpha}-helix of the EcRDBD with alanine (for more details see Fig. 1Go). The wild-type EcRDBD and alanine point mutants were overexpressed in Escherichia coli and purified to homogeneity (data not shown). The binding affinities of the DBDs were determined by EMSA using a double-stranded oligonucleotide containing the original hsp27pal sequence (Fig. 1BGo). As the hsp27pal had been shown previously to bind specifically and in a synergistic manner both the EcRDBD homodimer and the EcRDBD/UspDBD heterodimer (albeit with different affinities) (11), we tested the putative influence of alanine substitutions on homo- and heterodimer interaction. The effects of mutations on the EcRDBD homodimer binding are illustrated in Fig. 2AGo, and their quantitative analysis is presented in Fig. 2BGo. The binding of hsp27pal by homodimers was clearly reduced by alanine substitutions at positions G20, K22, R26, R27, S28, and K31. Among these mutants K22A, R26A, R27A, and K31A demonstrated the greatest DNA-binding defect, whereas the homodimerization of G20A and S28A mutants was impaired moderately. In contrast, alanine substitution of G23 and T30 increased affinity of the EcRDBD homodimer for the target sequence. Interestingly, the substitution of E19 by alanine did not appreciably alter the affinity of the EcRDBD. This result was unexpected as E19 corresponds to one of the three receptor-specific amino acids from the recognition {alpha}-helix, referred to as the P box (15). The P box residues have been defined as principal factors that underlie the discrimination of different response elements (16).



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  		Fig. 1. Schematic Diagram of the Macromolecular Components Used in This Study

A, Amino acid sequence of the wild-type D. melanogaster EcRDBD (42 ). The numbering is relative to the first Zn2+-coordinating cysteine of the DBD. Residues in the open circles correspond to the P box amino acids. Nine residues from the DNA-recognition {alpha}-helix (E19, G20, K22, G23, R26, R27, S28, T30, and K31, boxed) and eight residues from the T box region (R67 to E74) were substituted with alanine. Five amino acids from the DNA-recognition {alpha}-helix were excluded from the alanine scanning. In particular, C18, and C21, which are two of the eight absolutely conserved cysteines that coordinate two Zn2+ ions in nuclear receptor DBDs, and highly conserved F25, F26, V29 residues that, according to data published for other receptors including our observations concerning UspDBD (34 ), contribute to hydrophobic interaction that stabilizes the DBD structure. B, Sequences of the oligonucleotide probes used in EMSA (only one strand is shown). The sequence of the hsp27pal oligonucleotide is based on the natural 20E pseudopalindromic response element (marked with arrows) from the D. melanogaster hsp27 gene promoter (4 ). hsp27{Delta}R is the derivative of the hsp27pal sequence that contains only the left half-site of the hsp27pal (marked with arrow).

 


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  		Fig. 2. DNA-Binding Activities of the Mutants of the EcRDBD DNA-Recognition {alpha}-Helix

EMSAs were carried out with hsp27pal (A, B, E, and F), hsp27{Delta}R (C and D), and with the indicated homogenous EcRDBD (A, B, C, and D) or equimolar mixture of the respective EcRDBD and the wild-type UspDBD (E and F). Panels B, D, and F represent quantitative analysis of the EMSA data presented in panel A (lanes 2–11), panel C (lanes 2–12), and panel E (lanes 2–11), respectively, carried out using Fuji Film FLA-3000 Fluorescent Image Analyzer. The columns indicate mean values of three independent experiments and bars indicate SD values. The complexes formed by one DBD molecule are indicated by CI, and those originated from homo- or heterodimer are indicated by CII; F, free probe. The DBDs forming the respective complex are denoted by U for UspDBD, E for EcRDBD. The protein concentrations were: A, lanes 2–12, 240 nM of the indicated EcRDBD; lane 13, the same amount of the wild-type UspDBD; lane 14, 120 nM of each wild-type DBD; C, protein concentrations were as in panel A; E, lanes 2–11, 60 nM of the wild-type UspDBD and 60 nM of the indicated EcRDBD; lane 12, 120 nM of the wild-type UspDBD; lane 13, 120 nM of the wild-type EcRDBD.

 
Because our previous study demonstrated that the hsp27pal 5'-half-site exhibits substantially higher affinity toward the EcRDBD than the hsp27pal 3' half-site (11), one can assume that the above results can be ascribed to the interaction of the EcRDBD molecules with the high-affinity target half-site. To test this supposition EMSAs were carried out with the derivative of the hsp27pal containing only the left half-site (hsp27{Delta}R, see Fig. 1BGo). As shown in Fig. 2Go, C and D, the EcRDBD monomer binding pattern mirrored essentially that observed for the EcRDBD homodimer. Interestingly, the careful comparison of the binding patterns presented in Fig. 2Go, B and D, indicates that at least for two mutants (G20A and S28A) the magnitude of the EcRDBD homodimer affinity changes is higher than that observed for the monomer. This would indicate that G20 and S28 represent residues that are primarily involved in the binding of the 3'-half-site of the hsp27pal element or in the proper folding of the EcRDBD molecule bound to this part of the element in the EcRDBD homodimer.

To test the contribution of the DNA-recognition {alpha}-helix in the UspDBD/EcRDBD heterocomplex formation, we analyzed interaction of the wild-type UspDBD and the EcRDBD mutants with the hsp27pal sequence. The results presented in Fig. 2Go, E and F, reveal that only the R27 residue of the EcRDBD is indispensable for heterodimer formation. This contrasts with the data for the EcRDBD homodimer (and monomer) where mutations of two additional residues (K22 and R26) were detrimental for hsp27pal binding. Thus, it appears that in the context of the heterodimer the EcRDBD molecule better tolerates these substitutions. Moreover, the overall magnitude of the observed changes for the affinity of the heterodimers formed by the wild-type UspDBD and the E19A, G20A, G23A, S28A, and K31A mutants was lower than for the EcRDBD homodimer and the monomer (compare panel F of Fig. 2Go with panels B and D).

Together these observations may indicate that amino acid residues from the DNA-recognition {alpha}-helix of the EcRDBD display different hsp27pal binding characteristics in homo- and heterodimers. This may be also a reflection of the remarkable adaptability of the EcRDBD molecule when it forms a heterodimer with the UspDBD (see results below and Discussion).

Effects of Amino Acids Substitution in the DNA-Recognition {alpha}-Helix on the EcRDBD Structure
We used circular dichroism (CD) spectroscopy to determine whether the above-observed differences in the EcRDBD binding activity arose from loss of the particular amino acid residue (i.e. absence of specific contacts made by the residue) or from perturbation of the higher order protein structure induced by the mutation of the residue. Because the mutated amino acids are located in the region of the EcRDBD, which has an {alpha}-helical character (12), the far-UV CD spectra were recorded and analyzed. Far-UV CD spectroscopy is especially sensitive to the {alpha}-helical structure (17), which usually comprises a substantial fraction of the DBD fold of nuclear receptors. The far-UV CD spectrum of the wild-type EcRDBD shows minima at 222 and 206 nm (Fig. 3Go). The first minimum (at 222 nm) is characteristic for the CD spectrum of an {alpha}-helical structure in globular proteins. The minimum at 206 nm corresponds to the second band, which is also characteristic for an {alpha}-helix (at 208 nm) (17). The blue shift of the 206 nm minimum and its amplitude, which is larger in comparison with that of globular proteins, may arise from the contribution of conformation other than the {alpha}-helix. As shown in Fig. 3Go, C, E, G, and H, the CD spectra of K22A, R26A, S28A, and T30A mutants are essentially identical to the spectrum obtained for the wild-type EcRDBD (see also Table 1Go for the quantitative estimation of the secondary structure changes); therefore, results of the DNA-binding analysis for these proteins can be interpreted as a consequence of loss of contacts of particular amino acid residues with DNA (see Discussion). The intensities of the spectra recorded for other mutants are reduced relative to that of the wild-type EcRDBD, indicating either a decrease of the secondary structure content ({alpha}-helix, see Table 1Go), or a change in equilibrium between folded and unfolded molecules. This would indicate that functional effects observed after mutation of the E19, G20, G23, R27, and K31 residues could be interpreted either as a consequence of structural perturbation introduced in the wild-type EcRDBD or as a result of loss of particular amino acid residue-DNA contacts (see Discussion). Interestingly, among these residues only substitution of the R27 residue led to a substantial reduction of the EcRDBD DNA-binding affinity in the homo- and heterodimers. Simultaneously, G20A and especially G23A mutants, which along with R27A exhibited the greatest changes in the CD spectra, retained full ability to form heterodimers on the hsp27pal element.



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  		Fig. 3. Far-UV CD Spectra of the Mutants of the EcRDBD DNA-Recognition {alpha}-Helix

CD measurements of protein solutions (10.0 µM) were done with the Jasco J-715 spectropolarimeter. Spectra were recorded with the response time of 1.0 sec and with the data point resolution of 1.0 nm using a cuvette with 0.1-cm path length. Five scans were averaged to obtain smooth spectra. The solid circles represent the spectra of the wild-type EcRDBD; the open circles represent the spectra of the indicated mutant. [{theta}]MRE indicates mean residue ellipticity (in degrees x cm2 x decimoles–1).

 

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  		Table 1. Estimation of the Secondary Structure of the DNA-Recognition {alpha}-Helix Mutants

 
Effects of Amino Acid Substitutions in the T Box of the EcRDBD on the Interaction with the hsp27pal Element
The importance of the EcRDBD C-terminal extension (CTE) sequence for the interaction with the hsp27pal has been stressed previously by mutational experiments, which demonstrated that deletion of the EcRDBD region corresponding to the so-called A box (see Fig. 1AGo) abolished the EcRDBD ability to bind DNA and thus to form homo- and heterodimers on the hsp27pal element (11). The true functional role of the EcRDBD CTE, however, has yet to be fully appreciated because the crystallographic data allowed visualization of only seven A box residues exclusively in the EcRDBD-retinoid X receptor DBD (RXRDBD) and not in the wild-type (i.e. EcRDBD-UspDBD) complex (12). The remarkable conservation of the CTE sequence among all known EcRs suggests that this region may be crucial for the EcR function (see Fig. 9Go). The conservation is particularly evident in the N-terminal region of the CTE, corresponding to the so-called T box (18). With the exception of the Bombyx mori EcRDBD, amino acids located in these eight positions are absolutely conserved among all known EcRs (Fig. 9Go). Furthermore, analysis of the sequence data of other nuclear receptors indicates that the EcRDBD T box sequence does not exhibit any significant sequence similarity to other members of the superfamily (data not shown), which highlights its potential importance in response element discrimination. To investigate the role of the T box sequence for the EcRDBD function, we obtained in a homogenous form (data not shown) mutated EcRDBD derivatives, each containing a single alanine residue at one of eight positions in the T box region. EMSA experiments clearly show that only two mutants, P68A and E74A, display the same hsp27pal binding characteristics as observed for the wild-type EcRDBD, i.e. they form in a synergistic manner complexes consisting of two EcRDBD molecules (Fig. 4Go), and the affinity of the monomeric (CI) and dimeric complexes (CII) observed for the mutants are indistinguishable from that observed for the wild-type EcRDBD (Fig. 4Go, panels B and H, respectively). R67A and V71A showed reduced affinity to hsp27pal probe as monomers as well as dimers (Fig. 4AGo, 4EGo). The strongest DNA-binding defect was observed when the second of the T box prolines (P73) was replaced by alanine (Fig. 4GGo). Interestingly, in the case of the V72A the affinities of the monomer and the homodimer complexes were substantially increased compared with that of the wild-type EcRDBD (Fig. 4FGo).



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  		Fig. 9. Alignment of the C-Terminal Extension (CTE) Sequences of the DBDs

The alignment was done using ClustalX(1.81) (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/). Asterisk indicates positions that have a single, fully conserved residue; colon indicates that one of the following strong groups is fully conserved. Amino acid residue numbers are relative to the first Zn-coordinating cysteine. Regions corresponding to the so-called T box are boxed. Sequences were taken from SWISS-PROT (http://au.expasy.org/sprot/). Abbreviations used and accession numbers (in parentheses) are as follows: Dm, D. melanogaster (fruit fly) (accession nos.: EcR: P34021; Usp: P20153); Bm, B. mori (silk moth) (P49881); Ms, Manduca sexta (tobacco hornworm) (P49883); Ae, Aedes aegypti (yellow fever mosquito) (P49880); Lm, Locusta migratoria (migratory locust) (O97095); Tm, Tenebrio molitor (yellow mealworm) (O02035); Hv, Heliothis virescens (noctuid moth) (O18473); Chs, Chilo suppressalis (Q8MYA6); Chf, Choristoneura fumiferana (spruce budworm) (O77240); Cv, Calliphora vicina (blue blowfly) (Q9GPH1); Cc, Ceratitis capitata (Mediterranean fruit fly) (O76827); Lc, Lucilia cuprina (green bottle fly) (O18531); Cht, Chironomus tentans (midge) (P49882); Aal, Aedes albopictus (forest day mosquito) (Q9U3Y4); Aam, Amblyomma americanum (L.) (ixodid tick) (044338); hRXR-{alpha}, human RXR-{alpha} (P19793); hTR, human thyroid hormone receptor ß-1(P10828); hFXR_ß, human farnesoid X-activated receptor (Q96RI1–2 Q96RI1 splice isoform 2 of Q96RI1); hVDR, human vitamin D3 receptor (P11473); DHR38, probable nuclear hormone receptor HR38 from D. melanogaster (fruit fly) (P49869).

 


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  		Fig. 4. DNA Binding of the EcRDBD T Box Mutants to hsp27pal

Gel retardation experiments were performed with hsp27pal and increasing amounts of the indicated EcRDBD (A–H). The respective complexes were visualized by phospho imaging (insets), and their quantitative analysis is graphically depicted. Solid circles and solid triangles represent CII and CI complexes formed by the wild-type EcRDBD; the open circles and open triangles represent CII and CI complexes formed by the indicated mutant. Protein concentrations (in nM) in lanes 1–13 were: 0, 8, 16, 32, 60, 120, 200, 240, 400, 500, 600, 800, and 1000. The figure shows a representative experiment; similar results have been obtained in at lest four independent experiments. For more details, see text and the legend to Fig. 2Go.

 
Given the putative role of the EcRDBD T box sequence in forming not only the EcRDBD homodimers but also the EcRDBD/UspDBD heterodimers, we analyzed interaction between the wild-type UspDBD and all EcRDBD derivatives containing alanine substitution in the T box-related sequence. Similarly as it was described above for the DNA-binding {alpha}-helix mutants, changes in the EcRDBD affinity against hsp27pal are generally reflected in the UspDBD/EcRDBD-hsp27pal interaction (Fig. 5Go). However, the magnitude of the observed changes was considerably lower than for the EcRDBD homodimer, and with one exception (P73A), the T box of EcRDBD readily accepts substitutions of all amino acids, when the EcRDBD is complexed with the UspDBD. This observation, along with the CD spectroscopy data (see below) indicating that alanine substitution of some of the T box residues induces significant structural perturbations, suggests again that the EcRDBD molecule exhibits pliability in contact with its partner (UspDBD).



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  		Fig. 5. DNA Binding of the EcRDBD T Box Mutants in the Presence of Wild-Type UspDBD

Gel retardation experiments were performed with hsp27pal and increasing amounts of an equimolar mixture of the indicated EcRDBD with the wild-type UspDBD. Protein concentration (in nM) were: lanes 1–13: 0, 4, 8, 16, 30, 60, 100, 120, 200, 250, 300, 400, 500 of each DBD. Solid circles represent the wild-type UspDBD/EcRDBD heterodimer; open circles represent the complexes formed by the wild-type UspDBD with the indicated mutant. Other details are as in the legend to Fig. 4Go.

 
Effects of Amino Acids Substitution in the T Box on the EcRDBD Structure
Similarly, as in the case of DNA-binding {alpha}-helix mutants, we used CD spectroscopy to answer the question whether substitution of the amino acid residues from the T box region induces any structural changes in the EcRDBD. As shown in Fig. 6Go, three of the analyzed mutants (R67A, P68A, and E74A) exhibited very similar spectra to that of the wild-type EcRDBD (quantitative analysis of the T box mutants spectra is shown in Table 2Go). Two of these proteins (P68A and E74A) exhibited the same hsp27pal binding characteristics as observed for the wild-type EcRDBD. R67A bound the hsp27pal probe with reduced affinity either as EcRDBD homodimer or as UspDBD/EcRDBD heterodimer. CD spectra of four other mutants (E69A, C70A, V71A, and V72A) have reduced CD intensity compared with wild-type EcRDBD, indicating that, in the case of these proteins, alanine substitutions lowered the content and possibly altered distribution of the secondary structure elements (see Table 2Go). Only two of these mutants (V71A and V72A) exhibited substantial changes of the affinity in the functional tests, whereas two others (E69A, C70A) displayed slightly reduced affinity. Finally, CD spectrum of the P73A mutant highlights again potential distinctiveness of the P73 residue within the EcRDBD T box sequence. A comparison of the P73A and the wild-type EcRDBD spectra shows that substitution of the P73 with alanine enhances intensity of the spectrum, which reflects increase of the secondary structure content (see Table 2Go).



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  		Fig. 6. Far-UV CD Spectra of the EcRDBD T Box Mutants

Solid circles represent the spectra of the wild-type EcRDBD; open circles represent the spectra of the indicated mutant, respectively. [{theta}]MRE is mean residue ellipticity (in degrees x cm2 x decimoles–1). Other details are as in the legend to Fig. 3Go.

 

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  		Table 2. Estimation of the Secondary Structure of the T Box Mutants

 
Properties of the Drosophila EcRDBD Mutant Containing Amino Acid Residues Characteristic for the Bombyx T Box
As demonstrated in Fig. 9Go, an alignment of the EcRDBD sequences indicates that T box amino acid residues exhibit remarkable conservation among all analyzed EcR sequences. A surprising originality is the sequence of the Bombyx mori EcR where the nonconservative substitution of P73 with Q, and conservative substitution of V72 with I, can be observed. Interestingly, a P residue is present in the Bombyx sequence just downstream of the T box at position 75 and upstream of the putative A helix (see Fig. 7AGo). To investigate whether these differences between species influence the structural and/or functional properties of the T box region, a triple mutant of the Drosophila EcRDBD was obtained having amino acids at positions 72, 73, and 75 substituted with the respective residues from the Bombyx EcRDBD (Fig. 7AGo, residues underlined). As shown in Fig. 7BGo, one of the only characteristics of the mutant that differs from the wild-type Drosophila EcRDBD is its slightly reduced ability to form EcRDBD homodimers. The substitution of three Drosophila residues with the Bombyx counterparts has not resulted in any significant changes in the ability of the mutated EcRDBD to act as a partner for the UspDBD (Fig. 7CGo). Simultaneously, CD spectra of the mutant and the wild-type EcRDBD are virtually superimposable (Fig. 7DGo; see also Table 2Go). This suggests that no overall structural changes were introduced by the triple mutation. The above results suggest that amino acid substitutions characteristic for the Bombyx mori EcRDBD conserve the structural/functional integrity of the T box region, at least in terms of the DNA-binding affinity.



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  		Fig. 7. The Triple Mutant of the Drosophila EcRDBD

The triple mutant (V72I/P73Q/N75P) of the Drosophila EcRDBD (D. m.), containing at positions 72, 73, and 75 amino acid residues occurring in B. mori (B. m.) EcRDBD (A), was obtained in a homogenous form (data not shown). The hsp27pal-binding affinity of the V72I/P73Q/N75P mutant (open circles) was analyzed in the absence (B) and in the presence (C) of the wild-type UspDBD and compared with the affinity wild-type Drosophila EcRDBD (solid circles). Panel D shows CD spectra of the Drosophila EcRDBD and of the triple mutant. For more details see legends to Figs. 3Go and 4Go.

 
Flexibility of the EcRDBD
As demonstrated above, many of the EcRDBD mutants, including these with the defined structural changes, exhibit a surprising tolerance for alanine substitution because they are able to form efficiently complexes with hsp27pal in the presence of the UspDBD. This suggests the flexibility and adaptability of the EcRDBD that would allow molding of this protein around the DNA and the UspDBD. To determine whether this observation is reflected in the stability of the EcRDBD molecule, we performed chemical denaturation experiments using guanidine hydrochloride (GdmCl). Because EcRDBD does not contain tryptophan, the progress of unfolding was monitored using intrinsic tyrosine fluorescence. The results presented in Fig. 8AGo show that EcRDBD is an extremely unstable protein. Starting from even the lowest denaturant concentrations, a gradual increase in the fraction of the unfolded protein could be observed. To determine whether this denaturation profile is characteristic only for the Drosophila EcRDBD molecules, a chemical denaturation experiment was carried out for the overexpressed and purified B. mori EcRDBD. As shown in Fig. 8AGo, the denaturation profiles of both proteins are virtually superimposable, which suggests that structural liability seems to be a general feature of the EcRDBDs. Notably, the triple mutant (V72I/P73Q/N75P) described above exhibits the same denaturation characteristics (Fig. 8AGo). It has been recently reported that truncating the highly positively charged CTEs of DBDs of glucocorticoid and estrogen receptors increased stability of both proteins (19). To test if the stability of the EcRDBD is dependent on the C-terminal region, the denaturation profile was obtained for the EcRDBD mutant with the deleted C-terminal region (A box) [EcRDBD{Delta}A (11)] containing many positively charged amino acid residues. Figure 8AGo shows that the denaturation curve of this protein is very similar to the denaturation curve obtained for the wild-type EcRDBD. This, along with data obtained for other DBDs, would imply that the A box sequence itself does not destabilize the EcRDBD and that the EcRDBD instability is an inherent property of the core part of this protein. In contrast to the EcRDBD, the UspDBD is a more stable molecule. The UspDBD denaturation curve is clearly shifted to higher concentrations of denaturant (Fig. 8AGo) and exhibits a two-state transition, previously reported for DBDs of mammalian steroid receptors (19). The EcR and Usp DBDs display also dissimilar CD spectra (Fig. 8BGo), which reflect differences in the secondary structure content of both DBDs. As shown in Table 1Go, the EcRDBD is characterized by a lower amount of the {alpha}-helix structure and a higher amount of the ß-structure than the UspDBD in the DNA-free form.



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  		Fig. 8. Dissymmetry in the Molecular Properties of the EcRDBD and UspDBD

A, Chemical denaturation profiles of the DBDs. Chemical denaturation of the wild-type and mutated DBDs was monitored by fluorescence emission measurements under conditions described in Materials and Methods. The panel shows denaturation profiles for the following DBDs: wild-type Drosophila EcRDBD, filled circles; wild-type Bombyx EcRDBD, open triangles; triple mutant (V72I/P73Q/N75P) of the Drosophila EcRDBD, filled triangles; Drosophila EcRDBD that does not contain A box sequence, open circles; wild-type Drosophila UspDBD, filled squares. B, Comparison of the EcRDBD and the UspDBD CD spectra. CD spectra of the wild-type Drosophila EcRDDBD (solid circles) and UspDBD (open circles) were recorded at 10.0 µM concentrations under standard conditions (see Materials and Methods). C, Renaturation of DBDs. EMSAs were performed with hsp27pal and with the indicated wild-type D. melanogaster (D.m.) or B. mori (B.m.) DBDs. The panel shows results obtained for the DBDs that were incubated at 8.7 µM concentration on ice for 1 h with 4.0 M GdmCl and then diluted to obtain 0.1 M GdmCl concentration (denoted as renaturated DBD), or for the DBDs that were treated in the same manner except that GdmCl was completely omitted or present at 0.1 M concentration (denoted as 0.1 M GdmCl). The protein concentrations were: lanes 1–10, 218 nM of the indicated DBD; lanes 11–14, 109 nM of the indicated DBD. D, Quantitative analysis of the data presented in respective lines of panel C as indicated. The quantitative analysis was carried out using a Fuji Film FLA-3000 Fluorescent Image Analyzer. The columns indicate mean values of three independent experiments and bars indicate SD values.

 
To answer the question of whether or not the GdmCl-denatured Usp and/or EcR DBDs are able to return to active structure, refolding experiments were performed. The DBD samples incubated with 4.0 M GdmCl were diluted, and their hsp27pal binding affinities were analyzed and compared with the activity of the control samples (Fig. 8Go, C and D). The control samples contained the same concentration of the respective DBDs and the residual concentration of the denaturant as the diluted samples; however, DBDs in the control samples were not exposed to the 4.0 M GdmCl. As shown in Fig. 8Go, C and D (lanes 3 and 4), the UspDBD recovered at least 90% of the control sample activity. Slightly lower recoveries, i.e. 79% and 75%, were observed for homodimeric complexes of Drosophila and Bombyx EcRDBDs, respectively (Fig. 8Go, C and D, lanes 6, 7, 9, and 10, respectively). Interestingly, heterodimeric complexes, formed using nondenatured DBD and renatured partner, exhibited almost identical activity (ca. 90% of the activity of the control sample containing nondenatured DBDs), irrespective of the type of DBD used (Fig. 8Go, C and D, lanes 11–14).

Thus, in contrast to the UspDBD, the EcRDBD seems to be characterized by low stability. Nevertheless, even after denaturation-renaturation cycle the EcRDBD finds its unique native conformation, and this fact is manifested in the ability of the EcRDBD to form homo- and heterodimers on the hsp27pal element.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although molecular studies of the EcR and the Usp are much less extensive than those of vertebrate heterodimeric nuclear receptors, it is already clear that the EcR exhibits a unique combination of characteristics and thus holds an exceptional position among the family. For example, unlike the vertebrate steroid receptors, the EcR by itself cannot bind its ligand, 20E; it must heterodimerize with its partner, the Usp, for the stabilization of a ligand-binding conformation (2, 3). Another very intriguing feature of the EcR/Usp heterodimer is its preference for response elements arranged as highly degenerated imperfect palindromes with a single intervening nucleotide (4, 5, 6, 7, 8, 9). This attribute clearly distinguishes the EcR/Usp heterodimer from the vertebrate counterparts, which prefer inherently asymmetric DNA-binding sites composed of directly repeated half-sites for formation of the complexes characterized by the defined anisotropy (20). In contrast to the vertebrate DBDs, which form nonsymmetric head-to-tail heterocomplexes with RXRDBD on direct repeats, the EcRDBD/RXRDBD and EcRDBD/UspDBD complexes are organized on IR-1 element in a head-to-head manner, with the same fragments of the EcR and the Usp/RXR DBDs brought to the subunit interface (12). However, because the regions of the EcRDBD are distinct in sequence from their counterparts in the Usp/RXR, the dimer interface has a clear nonsymmetric character (12). This contrasts with the observation made in the case of vertebrate steroid receptor complexes with the inverted repeat elements, in which the head-to-head organized DBDs use identical residues to form perfectly symmetric interactions (20). Thus, the EcR receptor, which relies on both inverted repeat target and heterodimerization, has characteristics typical of both steroid and nonsteroid receptors and seems to be an evolutionary link between the vertebrate steroid and nonsteroid receptors (12).

Here we show how the individual amino acids, located in two particular EcRDBD regions, contribute to the specific interaction with the natural response element and to the stabilization of the EcRDBD molecule. In addition, the data indicate for the first time that the EcRDBD appears to be a protein with a high degree of intramolecular plasticity.

DNA-Recognition {alpha}-Helix
Recently published crystallographic analysis of the EcRDBD/UspDBD complex has identified the main hook residues within the DNA-recognition {alpha}-helix of the EcRDBD. It has been shown that these residues, consisting of E19, G20, K22, R26, and R27, form base-specific contacts with the 3'-half-site of the IR-1 element (12). Our results indicate that with one exception (E19), all these residues are also critical when the 5'-half-site of the natural pseudopalindromic element (hsp27pal) is used as a target. Two other residues (S28, K31), which were not identified as hook residues in the EcRDBD/UspDBD/IR-1 crystal, seem to be involved in some hsp27pal-specific contacts. Interestingly, the same residues were identified to play a key role in the EcRDBD homodimer formation. Our previous study has demonstrated that the 5'-half-site of the hsp27pal element exhibits substantially higher affinity against the EcRDBD than the 3'-half-site (11). Thus, it is quite possible that the results obtained for EcRDBD homodimer reflect primarily the influence of the respective mutations on the EcRDBD molecule interacting with the high-affinity 5'-half-site. Interestingly, two residues (G20 and S28) seem to be the main contact residues of the EcRDBD located on the hsp27pal 3'-half-site.

Results presented in this study demonstrate, for the first time, the different impact of the amino acid residues of the DNA-recognition {alpha}-helix on the EcRDBD interaction with DNA. The greatest DNA-binding defects were observed for three residues (K22, R26, and R27), which are very often involved in formation of the specific contacts of different DBDs with DNA (21). However, the pattern of hydrogen bonds between these residues and respective bases varies and is case specific. Here, simultaneous analysis of the EMSA and the CD data demonstrates that only K22 and R26 are pure contact residues, which are not involved in the stabilization of the DNA-recognition {alpha}-helix and possibly of the whole EcRDBD molecule. This is also true for S28, which has not been defined as a hook residue in the EcRDBD/UspDBD/IR-1 structure (12). All other residues important for effective interaction with the hsp27pal, including R27, are, to some extent, involved in stabilization of the EcRDBD molecule. Thus, the observed changes in EcRDBD binding activity cannot be solely attributed to the loss of the specific contacts, due to the mutation of the defined amino acid residue. Further experimental support by crystallographic and/or nuclear magnetic resonance spectroscopy studies is needed to clarify ultimately the involvement of these residues in the specific contacts with the natural, i.e. asymmetric elements. Of particular interest among all analyzed residues was G23. Its substitution by alanine led to the most pronounced change in the EcRDBD molecule. However, the EcRDBD readily accepts this change. This, along with other observations indicating that magnitudes of the affinity changes against DNA were lowered in the EcRDBD/UspDBD heterocomplex, suggests that the EcRDBD exhibits exceptional plasticity (see below).

T Box
Mutational studies and analysis of the crystallographic structures of the nuclear receptor DBDs bound to asymmetrical response elements have underlined the functional importance of the so-called CTE of the core DBD (22). The CTE sequence, consisting of the T box (18) and the adjacent {alpha}-helix (A box) (23), is a unique and characteristic structural element, which mediates for the particular receptor its dimerization on asymmetrical elements and significantly contributes to DNA-binding specificity. Whereas A box residues are mainly involved in the specific contacts with the response element, T box performs different functions simultaneously, including specific base pairing, forming of the dimer interface, and, most importantly, bringing of the A box helix in the correct position (21). Recently reported crystal structures of the vitamin D receptor DBD in complex with response elements from three different promoters support this hypothesis and provide further evidence that the T box together with the A box {alpha}-helix are characteristic elements defining the conformation of the CTE and thus response element discrimination (24).

We examined the effects of alanine substitutions for eight T box amino acid residues on DNA-binding activity and structure stability of the EcRDBD molecule. As shown in Fig. 9Go, with the exception of two positions in B. mori, these amino acids are absolutely conserved among all other EcRs. This conservation cannot be easily explained for two residues (P68 and E74), which, according to our data, are dispensable at least for DNA binding and stabilization of the structure of the EcRDBD molecule. Interestingly, the R67 seems to be a pure contact residue, not involved in the stabilization of the domain. Because, according to the crystallographic data obtained for the EcRDBD/UspDBD heterocomplex on IR-1 element, this residue has not been reported to be involved in any contacts with DNA (12), it is reasonable to assume that our observation is specific for the natural response element. All other analyzed residues show evidence of structural function as indicated by the CD spectra changes. However, in the case of V71A, V72A, and P73A mutants, this involves unambiguous changes in the DNAbinding affinity of the EcRDBD monomer and homodimer. These residues, together with C70, make up the hydrophobic core of the T box region and in the EcRDBD/UspDBD complex on IR-1 element are localized in this part of the EcRDBD where a dramatic change in the polypeptide chain direction is observed (12). Two consecutive turns are responsible for it: ß-turn, which includes C70, V71, V72, and P73; and {gamma}-turn, consisting of P73, E74, and N75. The results presented in this work highlight the distinctiveness of the P73 residue: its mutation has a detrimental effect on the DNA-binding affinity of the EcRDBD homodimers and exhibits the strongest effect on the heterodimer affinity. As can be concluded from the structural data obtained for the EcRDBD/UspDBD bound to the IR-1 element (12), the P73 residue is not involved in any interactions important for stabilization of the core DBD. Thus, it is conceivable that its main task is the proper folding of the EcRDBD CTE. Theoretically, the EcRDBD deprived of the A box contains all residues needed for the effective formation of the heterocomplex with the UspDBD on the response element. This can be inferred at least from the above mentioned structural data. However, biochemical data obtained for such EcRDBD derivatives have clearly demonstrated that the A box is indispensable for effective heterodimer formation (11). Furthermore, structural data obtained for only seven of 25 A box residues of the EcRDBD complexed with the RXRDBD on the IR-1 element suggest that the A box is directed to the 5'-half-site (12). Thus, one could speculate that this part of the EcRDBD is involved in some additional interactions, not seen in the crystal structure, protein-DNA, or protein-protein-DNA interactions. It is quite possible that P73, in which amide and carbonyl groups are involved in crucial hydrogen bonds of ß- and {gamma}-turn, is a key residue positioning the A box of the EcRDBD. A similar function could be ascribed to the V72 residue. According to the structure of the EcRDBD/UspDBD heterocomplex on IR-1 (12), the V72 lies outside of the DBD core. However, due to the interaction with the phosphate backbone, the V72 is locked in the defined position outside. The function of the V71 seems to be more complicated because, according to crystallographic data, this residue is involved in hydrophobic interactions with side chains of the core DBD residues, i.e. V29 and Y13. Unique properties of the V71, V72, and P73 residues is further supported by the results obtained for two residues (E69 and C70) that are located in the short {alpha}-helical region starting the T box sequence. Although their mutations are detrimental to the EcRDBD structure, only a minute effect on the homodimer binding could be detected.

The ultimate elucidation of the role of the EcRDBD CTE needs further experimental support. In particular, this would involve crystallographic analysis of the EcRDBD/UspDBD heterodimers complexed with the natural response elements, which, in contrast to the idealized IR-1 element, could promote proper, i.e. ordered, folding of the EcRDBD C-terminal sequence. Nevertheless, results presented in our paper clearly indicate that, in contrast to the vertebrate receptors, the EcRDBD C-terminal sequence is involved in the stabilization of this domain and possibly in the interaction with the specific DNA sequences.

Plasticity of the EcRDBD
As shown above, the EcRDBD exhibits unexpected mutational tolerance, which is particularly manifested when the DNA-binding affinity of the EcRDBD/UspDBD heterodimers is analyzed. At least in the case of the DNA-recognition {alpha}-helix, this property clearly distinguishes the EcRDBD from the UspDBD. It has been shown previously that the UspDBD affinity changes caused by the substitution of the DNArecognition {alpha}-helix residues by the alanine are directly reflected in the UspDBD/EcRDBD-hsp27pal interaction pattern (13). Our preliminary results indicate that this is also true for the UspDBD T-box alanine mutants (25). This striking difference in the structural adaptability, which has not yet been observed for any vertebrate nuclear receptor DBDs forming heterodimeric complex, can be explained by the different properties of both DBDs. In contrast to the UspDBD, the EcRDBD molecule seems to be less stable, which unveils the absence of cooperative unfolding transitions characteristic of a well-defined (i.e. globular) tertiary folding. This property, along with the previously published gel filtration data (26) indicating that the apparent molecular mass of the EcRDBD (17.4 kDa) is greater than the predicted value (13.5 kDa), suggests that the EcRDBD (or at least some fragments of this domain) is akin to intrinsically unstructured proteins (IUPs), which are characterized by an almost complete lack of the folded structure and an extended conformation with a high intramolecular flexibility. A prominent feature of IUPs is that they can adopt different structures upon different stimuli or with different partners, which enables their versatile interaction with different targets (27). This phenomenon termed "binding promiscuity" or "one-to-many signaling" (28) seems to exist also in the case of the EcRDBD. According to results obtained in our laboratory (29), the EcRDBD is able to interact on the hsp27pal element not only with the EcRDBD and the UspDBD but also with the DBD of the orphan nuclear receptor HR38 from Drosophila. The biological significance of the last observation is not clear, because it has been reported that the full-length EcR and the HR38 receptor are not capable of binding to the hsp27pal element (30). Interestingly, the isolated DBDs of both receptors can interact on this element effectively, which suggests that, under certain conditions, formation of the EcR-HR38 complex on DNA might be possible. Another unique functional faculty of the IUPs is that their open structures are largely preserved when they complex with their target, which provides for a disproportionate large binding surface and multiple contact points. The existence of the large DNA-binding surface in the EcRDBD and multiple contact points involved in the interaction have been suggested for the first time by mutational experiments (13). Recently published structural data of the EcRDBD/UspDBD complex with IR-1 element fully support this hypothesis and show that the EcRDBD footprint on DNA extends well beyond its own AGGTCA site to reach over both its 3'-flanking sequences and a large portion of the UspDBD half-site. In total, the EcRDBD footprint extends over 13 bp (12). The exact nucleotide sequence of the response elements affects not only the overall affinity of a receptor for its site but also influences its tertiary structure (31, 32). Therefore, site-specific DNA binding may be a trigger for an active intramolecular event that changes the shape of the DBD and in consequence of the whole receptor in a response element-specific way (31, 32). This could result in binding certain ancillary proteins also in a site-specific way. Because transcriptional regulation for a specific gene depends upon interactions of these proteins, the exact DNA sequence of the response elements may be the crucial regulatory factor. The EcRDBD/UspDBD heterodimer complexed with the respective response element seems to be the appropriate candidate for such sequence-dependent ancillary protein selection. The integral part of the heterodimer is the EcRDBD, which, due to IUPs-like properties, could easily accommodate DNA-induced changes in the secondary and tertiary structure. Indeed, comparison of the secondary structure content estimated using the CD spectra presented in this paper and the published crystal structure indicates clear changes in the secondary structure of the EcRDBD (see Table 1Go). Because the most reliable analysis of secondary structure content based on CD spectra can be performed for {alpha}-helices (17), we focus on this secondary structure. Thus, we note a clear gain of the {alpha}-helical structure after binding of the EcRDBD to the response element. In particular, the {alpha}-helix content increases from 20.5% in the EcRDBD in the DNA-free state to 24.4% or to 28.6% in the EcRDBD complexed with the UspDBD or the RXRDBD on DNA, respectively. Notably, the latter values, calculated using data available at the protein data bank, represent the minimal content of the {alpha}-helical structure. According to the published data (12), significantly higher values can be calculated, i.e. 29.4% and 34.5%, respectively. Interestingly, for the UspDBD only a small change in the {alpha}-helix content could be observed. As demonstrated by mutational studies, only 8 bp among 15 bp in the hsp27pal are indispensable for effective interaction with the complex formed by the full-length EcR and the full-length Usp (33). Other positions are not obligatory for heterodimerization, and as previously suggested, these positions may be used for the fine tuning of the EcRDBD/UspDBD structure and thus biological response (33, 34). Because even subtle structural changes in the DBD can have long-range functional consequences, it is possible that in the full-length EcR DNA binding could affect tertiary structure of other EcR domains. The EcR ligand-binding domain looks like a suitable candidate for such intramolecular cross-talk. According to mutational (35) and crystallographic studies (14), this domain is also characterized by extreme flexibility and adaptability, properties that allow molding of the domain around two completely different ligands, steroid and nonsteroid agonists. Thus, plasticity seems to be a general feature of the EcR molecule. One reason why EcR, the only known ligand-dependent nuclear receptor in insects, has gained such an unusual characteristic among all other nuclear receptors may be its key position in the mediation of the 20E signal transduction pathways. The insect hemolymph carries a wide range of endogenous ecdysteroids, some of which are only present at the specific stages during development (36). Mounting evidence indicates that alternate transcriptional pathways are driven by ecdysteroids other than 20E. Coordinate changes in ecdysteroid-regulated gene expression occur at several stages in the Drosophila life cycle at times when the 20E titer is known to be low (37). Given the reported biological activity of these ecdysteroids, it seems reasonable to expect that a single EcR molecule can mediate some of the diverse pathways. Due to the presence of the pliable ligand- and DNA-binding domains, EcR could adopt different, although ligand- and response element-specific, conformations; thus, in cooperation with specific cofactors and/or primary transcription factors, EcR could act as a universal and versatile factor controlling numerous ecdysteroid-dependent genes in a tissue- and gene-specific pattern.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Bacterial Strain and Plasmid Vector
The plasmid pGEX-2T (Amersham Biosciences, Freiburg, Germany) containing the lacIq gene was used for the expression of DBDs as fusions with Schistosoma japonicum glutathione-S-transferase (GST). For production of DBDs, Escherichia coli strain BL21(DE3)pLysS (Novagen, Schwalbach/Taunus, Germany) was used.

Construction of DBD-Expression Vectors; Site-Directed Mutagenesis
The construction of the expression plasmids for the wild-type Drosophila melanogaster EcR and Usp GST-DBDs (pGEX-2T-EcRDBD, pGEX-2T-UspDBD respectively) has been described previously (26). The expression plasmid for the wild-type B. mori GST-DBD was constructed analogously using the following primers: 5'-gcccggggatccGCACCTCGACAGCAAGAG-3' (sense) and 5'-gcccggggatccGTCTTCGACTGTGGTCGTA-3' (antisense).

PCR-based megaprimer mutagenesis protocol (38) was used to introduce new codons within the DNA region encoding the putative T box and the recognition {alpha}-helix of the EcRDBD. Plasmid pGEX-2T-EcRDBD (26) was used as a template. The sequence of the recombinant DNA fragments was verified by dideoxy sequencing.

Overexpression and Purification of the Wild-Type and Mutant Proteins
The expression of GST-DBDs and purification of the wild-type Drosophila UspDBD, EcRDBD, and its mutated derivatives, as GST-free proteins, were performed as described previously (26). The B. mori EcRDBD was isolated using a procedure elaborated for the Drosophila EcRDBD.

DNA-Binding Assays
EMSA was performed under conditions described previously (11). The indicated amounts of protein(s) and constant amount (femtomoles) of the appropriate 32P-labeled double-stranded oligonucleotide and 110 ng of poly(dI-dC) (Amersham Biosciences) were incubated for 30 min on ice in a final volume of 25 µl binding buffer [50 mM Na2HPO4, 100 mM NaCl, 1 mM 2-mercaptoethanol, 5 µM ZnCl2, 10% (vol/vol) glycerol (pH 7.8) at 22 C]. The DBD-DNA complexes were separated from the free DNA on 5% polyacrylamide gel in 0.25x Tris/Borate/EDTA. The gel was precooled to 4 C overnight, prerun at 160 V for 20 min, and, after applying samples, the electrophoresis was continued at 4 C for 30 min at 270 V and then for 240 min at 200 V. After that the gels were dried under vacuum at 80 C and exposed to Imaging Plates (Fuji Photo Film, Tokyo, Japan). Fluorescence signals were scanned with the Fuji Film FLA-3000 Fluorescent Image Analyzer (Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany). Scans were read at 50-µm resolution and 16-bit quantitative image accuracy and then analyzed using AIDA Bio-Package software (Raytest Isotopenmeßgeräte GmbH).

Circular Dichroism Spectroscopy
CD spectra were recorded using a Jasco J-715 (Jasco International, Japan) spectropolarimeter in 0.1-cm cuvettes thermostated at 20 C in 50 mM Na2HPO4 buffer (pH 7.8 at 22 C) containing 250 mM NaCl, 1 mM 2-mercaptoethanol, 5 µM ZnCl2, 10% (vol/vol) glycerol. Spectra were recorded with a response time of 1.0 sec and with 1.0-nm resolution. Each spectrum shown is the result of five spectra accumulated and averaged.

Chemical Denaturation
Denaturation profiles were monitored by fluorescence ({lambda}ex = 275 nm, {lambda}em = 305 nm, at 18 C) using the Fluorolog-3 instrument (Jobin-Yvon/Spex Horiba, Longjumean, France) and quartz cuvettes with 1.0-cm path length. The concentration of the proteins was 11.5 µM (9.0 µM for V72I/P73Q/N75P mutant). The concentrated stock GdmCl solution (7.0 M) was added to the protein samples in the phosphate buffer [50 mM Na2HPO4, 250 mM NaCl, 1 mM 2-mercaptoethanol, 5 µM ZnCl2, 10% (vol/vol) glycerol (pH 7.8) at 22 C]. To obtain desired denaturant concentration, the defined volumes of the samples were withdrawn from the incubation mixture, and corresponding volumes of the GdmCl solution were added to the mixture to maintain final volume of 500 µl. Each data point was calculated considering changes in protein and denaturant concentration.

Protein Concentration
The concentration of the purified proteins was determined spectrophotometrically at 280 nm using absorption coefficients calculated according to Gill and von Hippel (39).


   ACKNOWLEDGMENTS
 
We thank Dr. Luc Swevers and Professor Kostas Iatrou (Institute of Biology, National Center for Scientific Research "Demokritos," Athens, Greece) for the cDNA clone encoding the full length of B. mori EcR (pBluescript-SK+-BmEcR). We are grateful to Professor Jacek Otlewski (Institute of Biochemistry and Molecular Biology, University of Wroclaw, Poland) for giving us an opportunity to perform CD spectra measurements. In addition, we thank Professor Olaf Pongs and Dr. Dirk Isbrandt (Center for Molecular Neurobiology, Hamburg, Germany) for generous support.


   FOOTNOTES
 
This work was supported by Grant 3P04B 009 23 from the Polish State Committee for Scientific Research.

Present address for M.S.: Institut de Genetique et de Biologie Moleculaire, BP10142, 67404 Illkirch Cedex, France

Present address for I.G.: Department of Cell Biology, University of Geneva, 30 Quai Ernest Ansermet 1211, Geneva 4, Switzerland.

Abbreviations: CD, Circular dichroism; CTE, C-terminal extension; DBD, DNA-binding domain; EcR, ecdysone receptor; EcRDBD, EcR DNA-binding domain; 20E, 20-hydroxyecdysone; GdmCl, guanidine hydrochloride; GST, glutathione-S-transferase; hsp27pal, natural 20-hydroxyecdysone 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; IUP, intrinsically unstructured proteins; RXR, retinoid X receptor; RXRDBD, RXR DNA-binding domain; Usp, ultraspiracle protein; UspDBD, Usp DNA-binding domain.

Received for publication April 15, 2004. Accepted for publication June 2, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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