place in the case of the ecdysteroid receptor response element? �

Nuclear receptors are ligand-dependent transcription factors responsible for controlling differentiation, growth and development of higher eukaryotes. Three amino acids within the recognition alpha-helix of the DNA-binding domain of the nuclear receptors constitute the so-called "P-box" which determines response element specificity. In the ultraspiracle (Usp) protein, which together with EcR forms the heterodimeric ecdysone receptor, the P-box residues are E19, G20 and G23. Substitution of E19, the most characteristic amino acid for estrogen receptor-like P-boxes, with alanine showed that the mutation did not appreciably alter the affinity of the wild-type Usp DNA-binding domain (UspDBD(WT)) for a probe containing natural ecdysone response element (hsp27(wt)). Since in many cases E19 contacts a G/C base pair in position -4, which is absent in hsp27(wt), we analysed the interaction of UspDBD(WT), E19A and other P-box region mutants with the hsp27(wt) derivative which contains a G/C instead of an T/A base pair in position -4. UspDBD(WT) exhibited higher affinity for this element than for hsp27(wt). Moreover, a different interaction pattern of P-box region mutants was also observed. Thus we conclude that the E19 residue of UspDBD is not involved in any hsp27(wt) sequence-discerning contacts. However, substitution of the hsp27(wt) T/A base pair in position -4 with G/C generates target sequence with distinct functional characteristics and possibly with a new specificity. These results could serve as a basis for understanding the role of the presence of a T/A or G/C base-pair in the position -4 in the two types of ecdysone response elements found in nature.

tants was also observed.Thus we conclude that the E19 residue of UspDBD is not involved in any hsp27 wt sequence-discerning contacts.However, substitution of the hsp27 wt T/A base pair in position -4 with G/C generates target sequence with distinct functional characteristics and possibly with a new specificity.These results could serve as a basis for understanding the role of the presence of a T/A or G/C base-pair in the position -4 in the two types of ecdysone response elements found in nature.

20-Hydroxyecdysone (20E
) is a steroid controlling larval moulting, metamorphosis and reproduction in insects and other Ecdysozoa (Kozlova & Thummel, 2000;Sluder & Maina, 2001).The hormone acts via its receptor -a heterodimer of two proteins (Yao et al., 1992) -the products of the EcR (Koelle et al., 1991) and ultraspiracle (Oro et al., 1990) genes (EcR and Usp, respectively).EcR and Usp belong to the nuclear hormone receptor superfamily, which comprises ligand-dependent transcription factors with a characteristic domain structure (Evans, 1988).However, ecdysone receptor holds a unique position among other receptors.Although its ligand is a steroid, the functional ecdysone receptor is a heterodimer (Yao et al., 1992) unlike vertebrate steroid hormone receptors (Gehring, 1998).Moreover, it seems to bind its response elements only upon ligand binding (Thomas et al., 1993), which is not true of the receptors heterodimerising with the vertebrate retinoic acid receptor (RXR) (Torchia et al., 1998) -a Usp ortholog.The most conserved part of nuclear receptors is the DNA binding domain (DBD) with two characteristic zinc-binding modules (Freedman et al., 1988).Following the binding of the proper ligand, nuclear receptors interact with specific DNA sequences called response elements (REs).REs are derived from a common consensus sequence which during evolution was duplicated and modified, and now comprise two types of sequences -direct repeats, characteristic for heterodimeric receptors and palindromes, preferred elements for vertebrate steroid hormone receptors (Gronemeyer & Laudet, 1995).The duplicated structure of the REs is in consistence with the nature of the nuclear receptors often forming homo-or heterodimers upon binding their REs.The main structure responsible for the specific recognition of the proper RE is the a-helix of the first zinc-binding module of the DNA-binding domain (Renaud & Moras, 2000).It has been demonstrated that only a few amino acids within the a-helix are responsible for the recognition of the correct RE.Three of them, which constitute the so-called "P-box", are the most important ones for the mechanism of RE discrimination (Green et al., 1988;Mader et al., 1989;Nelson et al., 1999).
Here we demonstrate that mutation of E19, the most characteristic amino acid found in all receptors containing estrogen receptor-related P-box sequences (Umesono & Evans, 1989), did not appreciably alter the affinity of UspDBD and the UspDBD/EcRDBD heterocomplex for hsp27 element.It was unexpected since E19 forms the most comprehensive, structurally conserved and specificity-determining contacts in other receptors (Schwabe et al., 1993;Rastinejad et al., 1995;Zhao et al., 1998;Meinke & Sigler, 1999;Rastinejad et al., 2000;Zhao et al., 2000).In order to determine the basis for the insensitivity of UspDBD towards the mutation of its supposedly most critical P-box amino acid, we analyzed the interaction of wild type UspDBD (UspDBD WT ) and its E19A mutant with the hsp27 element and a derivative thereof containing a G/C base pair in position -4 instead of T/A.Our results indicate that the E19 residue of UspDBD seems to be not involved in any hsp27 element sequence-discerning contacts.Interestingly, natural selection tends to favor EcREs which similarly as the hsp27 element contain a T/A base pair in the position -4, although the substitution of the T/A base pair with G/C generates target sequence with higher affinity and distinct functional properties.
Overexpression and purification of the wild-type and mutant proteins.The expression of GST-DBDs and purification of GST-free wild type and mutated UspDBD, were performed as described previously (Niedziela-Majka et al., 1998;Grad et al., 2001).The designations of the respective mutant UspDBD, are based on the amino acid single letter code (e.g., E19A = Glu-19 ® Ala).
Protein concentration.Concentration of the purified proteins was determined spectrophotometrically at 280 nm using absorption coefficients calculated according to Gill and von Hippel (1989).

RESULTS AND DISCUSSION
The E19 residue of nuclear receptor DBDs with estrogen receptor-related P-boxes forms most the defined, structurally conserved and specificity-determining contacts with the response elements.In many receptors E19 accepts a hydrogen bond from the N4 of the cytosine of base pair G-4/C-4 and in many cases it contacts the adenine of the T-3/A-3 base pair through a water molecule and also takes part in other complex interactions responsible for the response element recognition (Schwabe et al., 1993;Rastinejad et al., 1995;Zhao et al., 1998;Meinke & Sigler, 1999;Rastinejad et al., 2000;Zhao et al., 2000) (see Table 1A).Surprisingly, the introduced mutation of E19 did not appreciably alter the affin-ity of UspDBD for a ds oligonucleotide containing hsp27 EcRE (hsp27 wt ) (Fig. 2, compare lanes 1-3 and 4-6).However, one notable feature of many EcRE elements, including hsp27 wt , is that a T/A base pair is present at their position -4 instead of a G/C (Riddihough & Pelham, 1987;Antoniewski et al., 1993;Antoniewski et al., 1994;Lehmann & Korge, 1995;Lehmann et al., 1997).To check if E19 exhibits a potential for binding to the -4 position, we substituted the T-4/A-4 base pair in hsp27 wt with G-4/C-4 to create hsp27 -4G .This substitution significantly increased the DNA binding affinity of UspDBD WT in comparison with its affinity to hsp27 wt (Fig. 2, compare lanes 1-3 and 7-9).Unexpectedly, the E19A mutant exhibited lower affinity for the hsp27 -4G probe than to the wild-type sequence (Fig. 3 compare 3A and 3B; Fig. 2, compare the ratio of CI U complexes intensity to the intensity of free DNA (F) in lanes 4, 5, 6 with lanes 10, 11, 12).The above data may indicate that the interaction between the E19 residue and the G-4/C-4 base pair provides a substantial change in free energy, which overcomes some unfavorable interactions between UspDBD and the hsp27 -4G element, which take place when E19 is absent, i.e. replaced by A. In contrast, removal of E19 did not reduce significantly the affinity of UspDBD for the hsp27 wt sequence (Fig. 2, compare lanes 1, 2, 3 and 4, 5, 6).Together, these results suggest that in addition to E19 some other amino acids make the hsp27 -4G se- A. Amino-acid sequence of wild type UspDBD (UspDBD WT ).The numbering in bold is relative to the first Zn-coordinating cysteine of the DBD, whereas numbers in parenthesis relate to the residue position relative to the N-terminus of full-length Drosophila melanogaster Usp (Oro et al., 1990).Residues in open circles correspond to the P-box amino acids; residues (19-31, except for C21) from the putative DNA recognition a-helix that were substituted with alanine are boxed.Note that three of the mutant DBDs (F25A, F26A and V29A) could not be analyzed as they were unstable during purification (Grad et al., 2001).B. Sequences of the oligonucleotides used in electrophoretic mobility-shift assays.The sequences of the ds oligonucleotides are based on the sequence from the D. melanogaster hsp27 gene promoter (Riddihough & Pelham, 1987;O¿yhar et al., 1991).hsp27 wt contains a 15-bp semi-palindromic EcRE -marked with the arrows.hsp27 -4G is hsp27 wt with base pair G/C in position -4; the exchanged nucleotide is underlined.The numbering convention in hsp27 wt was taken from previous study (Grad et al., 2001).For clearness of presentation only one strand of ds nucleotides is shown.quence-specific contacts, which do not take place when hsp27 wt is used as a target sequence.To test this hypothesis we analyzed the interaction pattern of hsp27 -4G with UspDBD mutants where individual amino acids of the putative recognition a-helix were substituted with alanine (see Fig. 1A).The results presented in Fig. 4 indicate that substitution of the G20, K22, G23 T28 and K31 residues (P-box region amino acids) of UspDBD results in different magnitudes of effect on DNA binding than it was observed for hsp27 wt (compare Fig. 4,lanes 3,4,5,8,10 and 13,14,15,18 and 20,respectively).No clear differences were observed, however, when other amino-acid residues were mutated (compare Fig. 4,lanes 6,7,9,and 16,17,19,respectively).Thus, we conclude that the E19 residue of UspDBD seems to be not involved in any hsp27 wt sequence-discerning contacts.In contrast, when a G/C base pair is present at the -4 position of EcRE, E19 creates (possibly Binding of wild type UspDBD (UspDBD WT ) (lanes 1-3 and 7-9) and E19A mutant (lanes 4-6; 10-12) to the relevant element -hsp27 wt (lanes 1-6) and hsp27 -4G (lanes 7-12) was studied.The proteins and their concentrations are indicated at the top.Monomeric complexes between UspDBD and DNA are indicated by CI U ; F, free probe.To estimate the relative binding activities compare the ratio of CI U complexes intensity to the intensity of free DNA in the respective lanes.EMSAs were performed with the respective elements -hsp27 -4G (A) or hsp27 wt (B) and increasing amounts of E19A UspDBD.Protein concentration (in nM) in lanes 1-10 was 0, 8, 16, 32, 60, 120, 240, 400, 600, 800. in co-operation with other P-box region amino-acid residues) protein-DNA contacts, which are distinct from those of hsp27 wt (summarized in Table 1).Notably, the same seems to be generally true for the hsp27 -4G interaction with the UspDBD/EcRDBD heterodimer (Fig. 5).These observations suggest that substitution of the hsp27 wt T-4/A-4 base pair with G-4/C-4 would generate a target sequence with distinct functional properties and possibly with a new specificity.In the case of 20E receptor, most naturally occurring elements have the base pair T-4/A-4 (Riddihough & Pelham, 1987;Antoniewski et al., 1993;1994;Lehmann & Korge, 1995;Lehmann et al., 1997).The reason of this selection is unclear since binding site selection experiments (Vögtli et al., 1998) have shown that an oligonucleotide with the base pair G-4/C-4 binds the ecdysteroid receptor with the highest affinity.The basis for the T-4/A-4 selection might be associated with the ability to differentiate higher order complexes formation on EcREs with either a T/A or a G/C base pair in position -4.As was shown previously, hsp27 wt serves not only as a target for the UspDBD/ EcRDBD heterocomplex but contains all the structural information necessary for the synergistic formation of the homodimeric EcRDBD complex (Niedziela-Majka et al., 2000).Our preliminary results indicate that the hsp27 -4G element binds EcRDBD with higher affinity than hsp27 wt , but the presence of the G/C base pair at the -4 position appears to exclude the cooperative formation of the EcRDBD homodimeric complexes (data not shown).We therefore suggest that substitution of the T-4/A-4 base pair of hsp27 wt with G/C might generate a more restrictive binding element, which possesses structural determinants that favor the binding of the UspDBD/EcRDBD heterodimer, but at the same time deter the binding of the EcRDBD homodimer.In contrast, a T-4/A-4-containing target (i.e.hsp27 wt ) would interact in a clearly synergistic manner either with the UspDBD/ EcRDBD heterodimer or with the EcRDBD homodimer (Niedziela-Majka et al., 2000).
Although it is widely, but not universally, accepted that the functional 20E receptor is the heterodimer of Usp and EcR, we hypothesize that subtle nucleotide differences in the EcREs could provide structural basis for the discrimination of the Usp/EcR heterodimer vs. the EcR/EcR homodimer.The implications of this are that both dimers may contact some regulatory elements, for example hsp27 wt , yet they may be elements that specifi- EMSAs were performed with hsp27 wt (lane 1-10) or hsp27 -4G (lanes 11-20) and with 120 nM of the UspDBDs (see Fig. 1A).Monomeric complexes between UspDBD and DNA are indicated by CI U ; F, free probe.Note that, although for clarity the figure presents results only for one chosen amount of each DBD, for each protein and response element the experiment with a complete range of concentrations was performed, same as in Fig. 3 (not shown).
Authors thank Ms Miros³awa Ostrowska for her excellent technical assistance.

Figure 1 .
Figure 1.Sequences of the macromolecular components used in this study.

Figure 3 .
Figure 3. Comparative DNA-binding of the E19A mutant of UspDBD to hsp27 -4G and to hsp27 wt .

Table 1 . Contacts of the E19 residue of known DBDs with bases of response elements.
Schematic representation of contacts between side chain of the E19 residue of DBDs with estrogen receptor-type P-box and REs based on the respective crystallographic data (A) and results presented in the study (B).In the left column the DBDs of respective receptors are denoted; in the case of dimeric structures the order of the proteins on the RE is kept.On the right the respective oligonucleotides used in the experiments with interactions with E19 marked are shown.W, water molecule.Underlined are half sites of REs, in italics the spacer between half sites.Note that additional contacts in the case of hsp27 -4G are possible.