©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Elk-1 ETS DNA-binding Domain (*)

(Received for publication, August 26, 1994; and in revised form, December 16, 1994)

Paul Shore (1) Louise Bisset (1)(§) Jeremy Lakey (1) Jonathan P. Waltho (2) Richard Virden (1) Andrew D. Sharrocks (1)(¶)

From the  (1)Department of Biochemistry and Genetics, the Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom and the (2)Department of Molecular Biology and Biotechnology, the Krebs Institute, University of Sheffield, Sheffield, S10 2UH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The ETS domain family of transcription factors is comprised of several important proteins that are involved in controlling key cellular events such as proliferation, differentiation, and development. One such protein, Elk-1, regulates the activity of the c-fos promoter in response to extracellular stimuli. Elk-1 is representative of a subgroup of ETS domain proteins that utilize a bipartite recognition mechanism that is mediated by both protein-DNA and protein-protein interactions. In this study, we have overexpressed, purified, and characterized the ETS DNA-binding domain of Elk-1 (Elk-93). Elk-93 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and purified to homogeneity from both the soluble and insoluble fractions using a two-column protocol. A combination of CD, NMR, and fluorescence spectroscopy demonstrates that Elk-93 represents an independently folded domain of mixed alpha/beta structure in which the three conserved tryptophans appear to contribute to the hydrophobic core of the protein. Moreover, DNA binding studies demonstrate that Elk-93 binds DNA with both high affinity (K approx 0.85 times 10M) and specificity. Circular permutation analysis indicates that DNA binding by Elk-93 does not induce significant bending of the DNA. Our results are discussed with respect to predictive models for the structure of the ETS DNA-binding domain.


INTRODUCTION

Families of transcription factors are defined on the basis of the primary sequence homology within their DNA-binding domains. Notable examples for which three-dimensional structures have been resolved include the helix-turn-helix (for review, see (1) and (2) ), the zinc fingers (for review, see (3) ), the helix-loop-helix(4, 5, 6) , and the b-Zip proteins(7) . However, structural features of a representative member from the ETS domain transcription factor family remain to be elucidated.

Members of the ETS domain family of eukaryotic DNA-binding proteins were originally identified by their primary sequence homology with the protein product of the ets-1 proto-oncogene(8) . A homologous sequence of approximately 86 amino acids comprising the ETS domain has since been identified within more than 30 proteins, many of which are transcription factors (for review, see (9) and (10) ). It has been demonstrated for several proteins that the ETS domain corresponds to the DNA-binding domain (for review, see Refs. 9 and 10). Indeed, several ETS domain proteins have been shown to bind DNA sequences of approximately 10 base pairs containing the core consensus sequence (C/A)GGA(A/T) known as the ets motif (for review, see (9) and (10) ). One particular feature, which is diagnostic of almost all ETS domains, is the presence of 3 conserved tryptophans spaced 18 residues apart. Secondary structure predictions by several standard methods (11, 12) suggest that the ETS domain shows no strong homology to any known DNA-binding motifs, although certain regions are predicted to be alpha-helical. However, others have used the predictive method of hydrophobic cluster analysis, which suggests that the structure of the ETS domain may be related to that of the DNA-binding domains of Myb and HMG(13) .

Members of the ETS family of DNA-binding proteins are expressed throughout the Metazoa in a variety of cell types(14, 15) . ETS domain proteins are involved in a range of important biological processes, including cell growth control, differentiation, and embryonic development. For example, the protooncogene products Ets-1 and Ets-2 are transcriptional activators implicated in T-cell activation and development(16) . Moreover, the ETS domain proteins Pok/Yan and PntP2 are transcription factors of the sevenless pathway involved in regulation of photorecepter cells during Drosophila eye development(17, 18, 19) .

The transcription factor Elk-1 (20) is an ETS domain protein that is involved in the transcriptional regulation of the immediate-early gene c-fos (for review, see (21) ). Elk-1 forms a ternary nucleoprotein complex with the serum response factor (SRF) (^1)and the c-fos serum response element (SRE)(22) . Three domains within Elk-1 were initially defined on the basis of amino acid homology (23) within a subgroup of ETS domain proteins that also includes SAP-1 (23) and ERP (24) (Fig. 1A). Subsequently, biochemical functions have been ascribed to each of these domains. The C-terminal region of Elk-1 contains a transcriptional activation domain regulated by growth factor-induced mitogen-activated protein kinases(25, 26, 27) . The Bdomain is a short internal region of 20 amino acids that is necessary for ternary complex formation(23, 28) . This domain mediates direct, specific, protein-protein interactions between Elk-1 and the DNA-binding domain of SRF(29) . The A-domain of Elk-1 comprises the ETS DNA-binding domain and is located at the N-terminal end of the protein. When in complex with SRF, the ETS domain of Elk-1 makes direct contact with the ets binding motif within the SRE; autonomous DNA binding of Elk-1 to the ets site within the SRE does not occur (28) . (^2)However, in the absence of SRF, Elk-1 specifically binds to the Drosophila E74 ets site (28) (^3)and other artificial sites (30) in vitro.


Figure 1: Purification of the ETS DNA-binding domain of Elk-1. A, schematic representation of the domain structure of Elk-1. The location of the A-domain (the ETS DNA-binding domain; amino acids 1-90), the B-domain (SRF interaction domain; amino acids 148-168), and C-domain (mitogen-activated protein kinase target/transcriptional activation domain; 352-399) are indicated. The fragment of Elk-1 representing the ETS DNA-binding domain of Elk-1 utilized in this study (amino acids 1-93; Elk-93) is shown below the full-length protein. B, SDS-polyacrylamide gel electrophoresis analysis of Elk-93 samples at various stages of purification; soluble and insoluble extracts from uninduced E. coli. harboring pAS74 (lanes2 and 3), soluble and insoluble extracts from E. coli. harboring pAS74 after 3 h of isopropyl-1-thio-beta-D-galactopyranoside induction (lanes4 and 5), soluble and insoluble protein samples after incubation of the insoluble induced extract shown in lane5 with 500 mM NaCl (lanes6 and 7), glutathione S-transferase-Elk-93 fusion protein after glutathione affinity chromatography (lane8), glutathione S-transferase (lane9), and Elk-93 (lane 10) after thrombin cleavage. Molecular mass markers are shown in lane1 with sizes in kDa indicated on the left. C, elution profile of Elk-93 after fast protein liquid chromatography heparin affinity chromatography. Elk-93 elutes as a single peak between 350 and 500 mM NaCl (fractions 9-12). 5-µl samples from 1-ml fractions 8-13 were analyzed by SDS-polyacrylamide gel electrophoresis (inset). Elk-93 in fractions 9-12 migrates as a single band with molecular mass approx11 kDa.



In this study, we have overexpressed the Elk-1 ETS domain as a fusion with glutathione S-transferase. A purification protocol has been developed to obtain homogenous preparations of the isolated ETS domain from both the soluble and insoluble cell fractions. A combination of biochemical and biophysical techniques has been used to establish that the purified isolated ETS domain is a functional, monomeric, DNA-binding protein with a dissociation constant of approx0.85 times 10M. We also demonstrate that the Elk-1 ETS domain binds DNA specifically and unlike many DNA-binding domains, does not appear to induce significant DNA bending as judged by the circular permutation assay. Circular dichroism (CD) and nuclear magnetic resonance (NMR) studies show that the ETS domain is a mixed alpha/beta structure. Furthermore, fluorescence spectroscopy indicates that the repeating tryptophan residues are buried within the interior of the protein. We discuss our results in relation to the various predictive structural analyses that have been carried out on the ETS DNA-binding domain.


MATERIALS AND METHODS

Plasmid Construction

pAS74

The sequence encoding the ETS domain of Elk-1 (Elk-93; amino acids 1-93) was amplified from pBsElk (encoding Elk-1; kindly provided by P. Shaw) using the polymerase chain reaction with the oligonucleotides ADS105 (5`-CGCTGGATCCGAATTCATCACCCTGCGACCTCAGGGTAG-3` and ADS106 (5`-CGACGGATCCATGGACCCATCTGTGACGC-3`). The product was cleaved with BamHI and EcoRI producing a 90-bp BamHI-EcoRI fragment and a 180-bp EcoRI fragment. The BamHI-EcoRI fragment was ligated into pGEX-2T (31) cleaved with BamHI and EcoRI to produce the plasmid pAS73. The 180-bp EcoRI fragment from the polymerase chain reaction was subsequently ligated into pAS73 to yield pAS74. The orientation of the inserted fragment was verified by restriction analysis. pAS74 encodes the N-terminal 93 amino acids of Elk-1 (representing the ETS domain) fused to glutathione S-transferase under the transcriptional control of the tac promoter.

pAS75

The plasmid pAS75 was constructed by ligating the phosphorylated, annealed oligonucleotides ADS107 (5`-CTAGAGCTGAATAACCGGAAGTAACTCAT-3`) and ADS108 (5`-CTAGATGAGTTACTTCCGGTTATTCAGCT-3`), into the Xbal site of the plasmid pBend2(33) . The inserted sequence was verified by DNA sequencing.

Expression and Purification of the Elk-1 ETS Domain

Escherichiacoli X-90 were transformed with the plasmid pAS74, and a single colony was used to inoculate 50 ml of ZB media (10 g of N-(Z)-amineA and 5 g of NaCl in 1 liter of water) containing 200 µg/ml of ampicillin. After overnight incubation at 37 °C with shaking at 200 rpm the culture was used to inoculate 1 liter of 2 times TY media (200 µg/ml ampicillin). Bacterial growth and subsequent induction of fusion protein expression was carried out essentially as described by Smith and Johnson(31) .

All steps in the purification, unless otherwise stated, were performed at 4 °C. Cells were pelleted by centrifugation, resuspended in 5 ml of PBS (16 mM Na(2)HPO(4), 4 mM NaH(2)PO(4), 150 mM NaCl, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and lysed by 6 times 30-s rounds of sonication on a MSE Soniprep 150 sonicator at an amplitude setting of 18 µm. The expressed fusion protein, glutathione S-transferase-Elk-93, was found in both the soluble and insoluble phases. The soluble protein was purified essentially as described by Smith and Johnson (31) with an additional high salt wash to selectively remove nucleic acid. Specifically, glutathione S-transferase-Elk-93 was bound to a 1-ml reduced glutathione-agarose column (Sigma) and washed with 10 column volumes of PBS to remove unbound protein. A second wash procedure with 20 column volumes of PBS containing 750 mM NaCl was then performed to specifically remove nucleic acids from the column. The presence of nucleic acids in the eluant was detected by spotting aliquots onto agarose plates containing 10 mg/ml ethidium bromide.

The insoluble protein was extracted from the insoluble pellet by resuspending the pellet in 10 ml of PBS containing 500 mM NaCl and incubating on a rotating drum for 1 hr. The protein suspension was centrifuged at 18,000 rpm for 20 min to remove insoluble material. The supernatant containing solubilized glutathione S-transferase-Elk-93 was then applied to a reduced glutathione-agarose column, and purification continued as described for the soluble fusion protein.

After thrombin cleavage (1 unit/ml; Sigma) the Elk-1 ETS domain (Elk-93) was eluted from the reduced glutathione-agarose column in 10 ml of TN buffer (50 mM Tris, pH 7.5, 150 mM NaCl). A 5-ml Econo-pac heparin cartridge (Bio-Rad) linked to fast protein liquid chromatography (Pharmacia) was used for the final purification step. The column was equilibrated with TN buffer prior to loading 10 ml of Elk-93 at a flow rate of 0.5 ml/min. The column was washed with 20 ml of TN buffer followed by a 20-ml linear gradient of NaCl (0.15-1 M) in 50 mM Tris, pH 7.5, at a flow rate of 1 ml/min. Column fractions of 1 ml were collected. Elk-93 eluted as a single peak between 350 and 500 mM NaCl.

The concentrations of protein samples were routinely estimated using the Bio-Rad protein assay reagent. A standard curve was derived using known concentrations of bovine serum albumin as recommended by the reagent supplier. Alternatively, sample concentrations were determined by ultraviolet absorption at 280 nm using the theoretical extinction coefficient of 24,750 M derived from the the Elk-93 primary amino acid sequence(32) . N-terminal protein sequencing was performed on an Applied Biosystems Inc., model 473A automated sequencer, at the Krebs Institute, Sheffield, UK. Samples were concentrated using Centricon microconcentrators (Amicon) with a molecular mass cut-off of 10 kDa according to the manufacturer's instructions.

DNA Binding Assays

Gel retardation assays were performed essentially as described previously(34) . Double-stranded oligonucleotide binding sites were constructed by annealing either the two oligonucleotides ADS155 (5`-GAATAACCGGAAGTAAC-3`) and ADS156 (5`-GGTTACTTCCGGTTATT-3`) to produce E74A or ADS107 and ADS108 (see ``Plasmid Constructions'') to produce E74B. E74A and E74B are identical except that E74B contains additional flanking sequences. The central ets motif is underlined in both sites. Double-stranded oligonucleotides were end-labeled using T4 polynucleotide kinase in the presence of [-P]ATP and subsequently gel purified. In standard reactions, P-labeled DNA binding sites (0.2 ng) were incubated with protein samples in a total volume of 12 µl containing 42 mM NaCl. Binding reactions were allowed to proceed for 20 min at room temperature prior to loading onto 5% nondenaturing polyacrylamide gels.

Competition experiments were performed by the addition of either unlabeled E74B oligonucleotide or unlabeled mutant E74 oligonucleotide mE74B (synthesised by annealing ADS125 (5`-CTAGAGCTGAATAACCGCAAGTAACTCAT-3`) and ADS126 (5`-CTAGATGAGTTACTTGCGGTTATTCAGCT-3`) in the reaction mixture prior to the addition of P-labeled E74B probe; reactions were allowed to proceed for an additional 20 min prior to loading onto the gel.

Gels were dried, and DNA-protein complexes were visualized by autoradiography. Quantification of DNA-protein complexes was achieved by phosphorimaging (425 S PhosphorImager and ImageQuant software; Molecular Dynamics).

DNA bending analysis was performed on circularly permuted 145-bp DNA fragments containing the E74B binding site obtained from the plasmid pAS75. Fragments were routinely labeled with P using the kinase exchange reaction (35) or alternatively synthesized by polymerase chain reaction in the presence of [alpha-P]dCTP.^3

The apparent dissociation constant (K(d)) for Elk-93 was obtained using gel retardation analysis in which the protein concentration was held constant (2.3 times 10M) while the DNA concentration was varied (0.15-1.04 times 10M). Under these conditions, the dissociation constant can be calculated graphically by subjecting the data to Scatchard analysis. K(d) is defined from the gradient of the resulting graph representing the equation; [PD] = -K(d)[PD]/[D] + [P(t)], where [D] is the concentration of free DNA, [PD] is the concentration of protein:DNA complexes, and [P(t)] is the concentration of total active protein. DNA binding reactions were set up under standard conditions except that they were carried out at 125 mM KCl in the absence of poly(dIbulletdC) competitior. Dilutions of Elk-93 were carried out in SDB buffer (1 times PBS, 10% glycerol, 1 mg/ml bovine serum albumin).

Circular Dichroism

Circular dichroism spectra were collected at 20 °C in a Jobin-Yvon CD6 spectrometer calibrated using 10-(camphorsulfonic acid). Quartz cells of 0.1 mm were used for measurements in the far ultraviolet (195-250 nm). Mean residue values were calculated using the predicted protein sequence and concentrations derived from the absorption measurements at 280 nm on a Cary 4E absorption spectrometer. The samples contained 50 mM Tris buffer, pH 7.4, and 470 mM NaCl. Contin analysis used the full 16-protein spectral data set(36) .

NMR Spectroscopy

Samples for NMR spectroscopy were prepared in 150 mM PBS, pH 7.4, containing 10% D(2)O. Experiments were performed at 298 K. Both one- and two-dimensional NMR spectra were recorded with presaturation of the water resonance (1 s) and Hahn-echo detection (37) with a pre-echo delay of 2.54 ms. Each dataset was subjected to a time domain convolution difference filter (38) using data acquired prior to the spin-echo to remove the residual water resonance(39) . Total correlated spectroscopy (TOCSY) experiments (40, 41) were recorded with DIPSI-2 spin-locking of longitudinal magnetization for 30 ms at a field strength of 10 kHz. Data were processed using the program FELIX (Biosym, CA). Phase-shifted squared sine-bell window functions were applied in each dimension prior to Fourier transformation.

Fluorescence Spectroscopy

Steady-state fluorescence emission spectra of Elk-93 and N-acetyl-tryptophanamide were measured in an Edinburgh instruments FS-900 fluorimeter. All measurements were made in a 1-cm path length quartz cuvette at 25 °C with both excitation and emission bandwidths of 4 nm. The excitation wavelength used was 295 nm. The concentrations of Elk-93 and tryptophanamide in 50 mM Tris buffer, pH 7.4, 150 mM NaCl were 20 µg/ml and 30 µg/ml, respectively. Denaturation of Elk-93 was carried out in the same buffer containing 6 M guanidinium chloride with incubation for 18 h at 25 °C. Sample solutions having an absorbance of less than 0.1 were used to minimize the inner filter effect. All spectra were corrected for the wavelength dependence of the instrument response.


RESULTS

Purification of the ETS Domain of Elk-1

A 93-amino acid polypeptide comprising the ETS DNA-binding domain of Elk-1 (Elk-93) (Fig. 1A) was expressed in E. coli as a fusion protein with glutathione S-transferase (glutathione S-transferase-Elk-93). Elk-93 was purified from isopropyl-1-thio-beta-D-galactopyranoside-induced E. coli X90 cells harboring the expression plasmid pAS74. pAS74 encodes the glutathione S-transferase-Elk-93 fusion protein that, upon induction, represents approximately 30% of the total cell protein (Fig. 1B, lanes4 and 5). SDS-polyacrylamide gel electrophoresis analysis of the soluble and insoluble protein samples revealed that approximately 80% of the induced fusion protein was present in the insoluble fraction (Fig. 1B, lanes4 and 5). It has previously been demonstrated that in the case of the DNA methyltransferase M.HhaI(42) , insoluble protein could be solubilized in a high salt buffer. The insoluble protein from the glutathione S-transferase-Elk-93 preparation was resuspended in phosphate buffer containing 500 mM NaCl. After two rounds of salt extraction, approximately 60% of the glutathione S-transferase-Elk-93 fusion protein was present in the soluble phase (Fig. 1B, lanes6 and 7). Subsequent extractions failed to further solubilize significant amounts of protein,^2 indicating that this was possibly of a more insoluble nature such as in inclusion bodies (Fig. 1B, lane7).

The solubilized high salt fractions were pooled and bound to reduced glutathione-agarose beads; binding to the beads was not prevented by the high salt concentration (Fig. 1B, lane8). After unbound proteins were removed by washing, an additional step was introduced to selectively remove nucleic acids associated with the fusion protein. PBS containing 750 mM NaCl was used to wash the column in order to selectively elute nucleic acids from the column while retaining the protein. After thrombin cleavage, the ETS domain was eluted in 2 column volumes of TN buffer (Fig. 1B, lane10). In order to remove the thrombin and achieve further purification, the Elk-93 ETS domain was applied to a heparin column and eluted with a linear NaCl gradient (Fig. 1C). Elk-93 elutes between 350 and 500 mM NaCl. This two-column purification yielded approx0.6 mg of pure Elk-93/liter of bacterial culture. SDS-polyacrylamide gel electrophoresis analysis of the purified protein demonstrates that the recombinant purified Elk-93 ETS domain migrates as a single band of molecular mass approx11 kDa; the preparation was judged to be homogeneous (>98%) by Coomassie staining. Elk-93 from the original soluble fraction was bound to reduced glutathione-agarose beads and purified by the same protocol after thrombin cleavage^2 and yielded approx0.2 mg/liter. The total yield from the soluble and insoluble phases was approx0.8 mg/liter. N-terminal sequencing of the purified polypeptide demonstrated that the first 2 residues were glycine and serine from the glutathione S-transferase moiety; this is expected after thrombin cleavage. The next 10 amino acid residues were identical to the predicted sequence for the Elk-1 ETS domain.

DNA Binding Properties of Elk-93

The purified Elk-93 protein specifically recognizes the ets motif within the E74 binding site. A gel retardation assay was performed in which a mutated E74 binding site with a single base change within the core GGA motif (mE74B) was used as a competitor (Fig. 2A). Competition with increasing amounts of the unlabeled wild-type E74B binding site reduced binding at all concentrations and abolished binding at 100-fold molar excess (Fig. 2A, lanes8-13). In contrast, increasing amounts of the mutant mE74B competitor did not prevent binding of Elk-93 to the wild-type E74B DNA even when present in 200-fold molar excess (Fig. 2A, lanes2-7), clearly demonstrating the specificity of DNA binding. This binding specificity appears identical to that derived using longer in vitro translated Elk-1 derivatives (28) .^2


Figure 2: DNA binding properties of Elk-93. A, DNA competition assay by gel retardation analysis demonstrating specific DNA binding of Elk-93 to the E-74 site. Elk-93 was incubated with P-labeled E74B oligonucleotide in the presence of increasing amounts of either mutant (mE74B; lanes2-7) or wild-type (E74B; lanes8-13) oligonucleotides. The amounts of competitor DNA included in the binding reactions are; 0 ng (lane1), 0.2 ng (lanes2 and 8), 2.5 ng (lanes3 and 9), 5 ng (lanes4 and 10), 10 ng (lanes5 and 11), 20 ng (lanes6 and 12), and 40 ng (lanes7 and 13). The increase in competitor concentration is indicated schematically above each series of lanes. B, circular permutation analysis of Elk-93 binding to the E74B site. The location of the E74B binding site (represented by a hatchedrectangle) within the 145-base pair circularly permuted probes is shown schematically on the right. Probes were made by cutting pAS75 with the following restriction enzymes; BamHI (Ba), PvuII (Pv), MluII (Ml). The result of the gel retardation experiment is shown on the left. DNA binding sites were; Ba (lane1), Pv (lane2), Ml (lane3). The location of protein:DNA complexes (PD) and free DNA (D) are indicated. C, gel retardation analysis of Elk-93 binding to the E74A oligonucleotide in the presence of increasing concentrations of NaCl. The percentage of maximal DNA binding by Elk-93 at each NaCl concentration was determined by phosphorimaging and is shown graphically. The inset shows the results of the gel retardation experiment. NaCl concentrations in each binding reaction are; 0 mM (lane1), 42 mM (lane2), 83 mM (lane3), 167 mM (lane4), 333 mM (lane5), 500 mM (lane6), 667 mM (lane7), and 833 mM (lane8). The increase in NaCl concentration in each reaction is indicated schematically above the gel.



It has been demonstrated that several transcription factors induce bending in the DNA sequence upon binding(43, 44, 45, 46) . Since SRF induces bending in the SRE(47) ^3 we investigated whether Elk-93 was also capable of bending DNA. Such bending may contribute to a possible mechanism of transcriptional regulation at Elk-1 targets such as the c-fos SRE. DNA bending can be analyzed using the circular permutation assay(48) . This assay is based on the observation that the mobility of a DNA fragment through a nondenaturing polyacrylamide gel is dependent upon the extent to which the fragment is bent. Bending at the center of a DNA fragment results in a lower mobility than if the bend occurs at one end. Three circularly permuted DNA fragments of identical size were produced that contain the E74B binding site located either at the center or toward the ends of the fragment (Fig. 2B). All three fragments when bound by Elk-93-produced complexes that migrated with virtually identical mobilities in a 5% nondenaturing polyacrylamide gel (Fig. 2B, lanes13). These results indicate that the isolated ETS domain of Elk-1 does not significantly bend the E74B DNA sequence.

The effect of increasing salt concentrations on the DNA-binding activity of Elk-93 was also investigated. Elk-93 binds the E74A site in the absence of NaCl and remains bound at all NaCl concentrations tested between 42 mM and 833 mM (Fig. 2C). DNA binding appears to be relatively insensitive to high NaCl concentrations, indicating that the protein is stable in high ionic strength solutions.

Elk-93 also binds the E74 site with high affinity. Gel retardation experiments were carried out in which the concentration of protein was held constant while the concentration of the DNA substrate was varied. The data from this experiment were subjected to Scatchard analysis (Fig. 3). An apparent dissociation constant, K(d), for Elk-93 of 0.85 times 10M was calculated from the gradient of the resulting graph. The value of the y axis intercept indicates that less than 10% of the protein was active in DNA binding. Such fractional binding activity is a poorly understood phenomenon(49) . However, Elk-93 purified from either the soluble or insoluble fractions binds with equivalent affinities and are defined spectroscopically as homogenous populations.^2


Figure 3: DNA binding affinity of Elk-93. Inset, results of gel retardation analysis in which the concentration of E74A oligonucleotide was titrated against a constant concentration of Elk-93 (2.3 times 10M). DNA concentrations were 0.15 nM (lane1), 0.3 nM (lane2), 0.45 nM (lane3), 0.6 nM (lane4), 0.75 nM (lane5), 0.9 nM (lane6), 1.04 nM (lane7). The increase in input DNA concentration ([D(t)]) is represented schematically above the gel. The locations of protein:DNA complexes (PD) and free DNA (D) are indicated. The results are represented by a Scatchard plot. The apparent dissociation constant for Elk-93 binding to the E74 binding site was calculated from the gradient of the resulting graph as 0.85 times 10M.



CD and NMR Analysis Demonstrate That Elk-93 Consists of a Mixed alpha/beta Structure

Both CD and NMR analysis were performed on the purified recombinant Elk-93 protein. The far ultraviolet CD spectrum for Elk-93 is shown in Fig. 4and is characteristic of a mixed alpha/beta structure such as flavodoxin or subtilisin(50) . The peak at 208 nm indicates the presence of alpha-helical secondary structure. Estimates of alpha-helical content ranging between 36 ± 2.2% and 43 ± 1.3% were obtained using the CONTIN program in the spectral ranges 195-240 to 200-240 nm. Approximately 30% beta-sheet is predicted. However, the correct assignment of beta-structure in this range is mathematically underdetermined (36, 51) and thus the relative amounts of unstructured versus beta-structure in the remaining 60% cannot be accurately predicted. Nevertheless, the similarity with known spectra (50) and lack of random coil signal (due to the correct zero value at 203 nm) makes the CONTIN prediction of beta-sheet content reasonable.


Figure 4: Circular dichroism spectra of the expressed domain. Elk-93 was measured at a protein concentration of 77 µM in a 0.1-mm cell. 20 scans were accumulated between 195 and 250 nm. The spectrum was corrected by subtraction of a blank containing only buffer. The abscissa is represented in units of Delta (M cm) using the molar concentration of amino acids present.



The one-dimensional NMR spectrum of Elk-93 (Fig. 5A) shows resonance line widths characteristic of a monomeric 11-kDa protein that is free from aggregation effects. The considerable dispersion of resonances indicates the independence of this domain as a folded protein. The two-dimensional TOCSY spectrum of Elk-93 (Fig. 5B) showing the ``fingerprint region,'' confirms the folded nature of the peptide backbone. This region of the TOCSY spectrum shows a high degree of dispersion including, in particular, a number of resonances with both downfield amide and alpha proton chemical shifts typically observed from residues in beta-sheets of proteins. A statistical analysis of alpha proton chemical shifts in proteins reveals that those of residues in coil conformations normally resonate in the region 4.1-4.6 ppm, whereas those of residues within beta-sheets resonate between 4.6 and 5.1 ppm(52) . In Elk-93, 23 residues have alpha proton resonances at frequencies lower than 4.6 ppm, and from this an estimate of 25 ± 7% beta-sheet can be derived. The size of the error reflects an estimate of other potential influences that may be occurring on the chemical shift of these resonances as seen in a wide range of proteins (for review, see (53) ). Although an analogous estimate of the helix content is less accurate, owing to the greater overlap of the chemical shift range with that of residues in coil conformations(52) , from the number of alpha protons resonating above 4.0 ppm it is likely that the ETS domain has a mixed alpha/beta-structure. These data are entirely consistent with results obtained using CD spectroscopy, which strongly predicts a high proportion of alpha-helical content and also indicates the presence of a significant proportion of beta-sheet.


Figure 5: ^1H NMR spectra of Elk-93. A, one-dimensional spectrum recorded with presaturation and removal of the residual water resonance with a time-domain convolution difference filter. B, fingerprint region of a 30-ms TOCSY spectrum showing the high degree of dispersion of the amide-alpha cross-peaks.



The Repeating Tryptophans Are Buried within the Interior of the ETS Domain

The 3 repeating tryptophan residues are present in almost all ETS domains. This high conservation suggests a fundamental role for these residues. Two such roles might be in either forming a structural scaffold or alternatively in DNA binding. Among protein sidechains, the fluorescence properties of tryptophan are uniquely sensitive to their local chemical environment. We therefore utilized fluorescence emission spectroscopy to determine whether these residues are solvent exposed or buried within the Elk-1 ETS domain.

The fluorescence emission spectra of native and denatured Elk-93 were compared with that of the model compound N-acetyltryptophanamide (NATA) (Fig. 6), which has a completely solvent-exposed indole side chain in aqueous solutions. The position of the peak in the NATA spectrum arises from the effects of solvent relaxation in which excited state energy is transferred to the aqueous phase prior to fluorescence emission. The position of the emission peak of Elk-93 is blue shifted relative to that of NATA. This indicates shielding of the tryptophan indole rings from general solvent effects by the bulk structure of the protein and is typical of a protein with buried tryptophan residues.


Figure 6: Corrected fluorescence emission spectra of Elk-93 and NATA. The excitation wavelength was 295 nm. Both native and denatured Elk-93 spectra are shown for comparison with the NATA spectrum. The native Elk-93 spectrum has a peak at 332 nm compared with the denatured Elk-93 and NATA spectra which peak at 350 and 352 nm respectively.



The emission spectrum of denatured Elk-93 is similar to that of NATA as expected for tryptophan residues that are fully exposed to the solvent (54) . Taken together, these data suggest that the tryptophan residues in the native Elk-1 ETS domain are buried within the interior of the protein and are likely to play a key structural role.


DISCUSSION

The structure of the DNA-binding domains of several eukaryotic transcription factors have recently been elucidated both in isolation and in DNA-bound complexes. The molecular interactions of transcription factors with particular DNA sites are of paramount importance to an understanding of the complex mechanisms of transcriptional regulation.

The ETS family of transcription factors is a large and important group of proteins that regulates the transcription of a wide range of genes. Both protein-DNA and protein-protein interactions are important in imparting the specificity of interactions of ETS domain proteins with their target promoters (for review, see (9) and (10) ). However, to date, there have been no structural studies on the DNA-binding domain of a representative member of the ETS family. Such information would provide the basis to our understanding of the specificity determination by protein-DNA interactions that are mediated by the ETS domain. We have therefore overexpressed and purified the ETS DNA-binding domain of Elk-1 (Elk-93) and subsequently characterized this domain using a plethora of spectroscopic and biochemical assays.

A simple two-column purification of the ETS domain has been developed that yields highly purified preparations of Elk-93. The purification scheme incorporates steps that solubilize the insoluble glutathione S-transferase-Elk-93 fusion protein by eluting in a high salt buffer. Such a strategy has previously been used to purify the DNA methyltransferase M.HhaI(42) . In addition, we have demonstrated that contaminating DNA, which is bound with the protein on the reduced glutathione-agarose column, can be specifically removed by a 750 mM NaCl wash. Purified proteins from both the soluble and insoluble fractions were identical as determined functionally by gel retardation analyses and spectroscopically by CD and NMR analyses.^2 Our protocol for purifying insoluble protein may be applicable to other DNA-binding domains expressed as fusion proteins with glutathione S-transferase.

The purified isolated Elk-1 ETS domain, Elk-93, binds the E-74 site with both high specificity and affinity with an apparent dissociation constant (K(d)) of 0.85 times 10M. This dissociation constant is within the same range as observed for a truncated version of Ets-1 containing the ETS domain (DeltaN322; (55) ). In this case, K(d) values varied between 0.4 and 4 times 10M depending on the DNA binding site. Moreover, this binding is stable in concentrations of NaCl up to 833 mM. This indicates that the function of Elk-93 is not greatly affected by the presence of NaCl and as such we conclude that our structural data obtained in high NaCl concentrations is likely to reflect the native conformation of the ETS domain. The salt insensitivity of DNA binding by Elk-93 is in marked contrast to that of other DNA binding proteins that lose their ability to bind DNA with high affinity and specificity at high salt concentrations. Notable examples include two members of the MADS family of transcription factors, SRF and RSRFC4/MEF2A, whose DNA binding is severely compromised in 250 mM KCl (56) and the estrogen receptor that shows greatly reduced DNA binding in 250 mM NaCl(57) . This may reflect a novel type of interaction between the ETS domain and DNA, which presumably is not highly dependent on salt bridges to the phosphate backbone that would be disrupted under high ionic strength conditions(58) .

Several DNA-binding proteins that interact with DNA via alpha-helices have been shown to bend DNA. Examples include CAP(48) , Fos/Jun(59) , the POU domain(43) , and more recently Myb(46) . Circular permutation analysis demonstrates that the ETS domain of Elk-1, while capable of specific binding to the E74 site, does not induce significant bending in this DNA sequence. The lowest limit to the sensitivity of the circular permutation assay is not known, although bend angles between 12 and 21° have been reported(60) . It is therefore possible that Elk-93 may introduce a low degree of bending into DNA that is not detectable by this assay. However, the apparent lack of DNA bending by Elk-93 detected by circular permutation analysis is in contrast to results using the same assay that demonstrates that the Myb DNA-binding domain induces significant (110-116°) DNA bending(46) . Myb contains 3 tryptophan residues with similar spacing to those found in the ETS domain. Based on the presence of these 3 conserved tryptophan residues, it has been postulated that the Myb and ETS DNA-binding domains are structurally related(13) . However, as the ETS domain is incapable of bending DNA, and indeed recognizes completely different sites than Myb, the mechanism of DNA binding is likely to be fundamentally different. Therefore, it is likely that the two domains will show a high degree of structural divergence. However, our results suggest that the 3 conserved tryptophan residues may play a similar role in the two DNA-binding domains. In Myb, these tryptophans are buried within the interior of the protein and form a hydrophobic core around which alpha-helices are packed(61) . Our fluorescence studies on the ETS domain containing similarly spaced tryptophans also indicate that these residues are within the interior of the domain. These highly conserved residues within the ETS domain may therefore play a structural role similar to that observed in Myb. Due to their buried nature, it is unlikely that these residues make direct contacts with DNA. Moreover, no change in fluorescence quenching by specific DNA is observed, (^4)further suggesting that these residues are not involved directly in DNA binding.

CD and NMR analysis have been used in this study to determine the nature of the secondary structure within the ETS domain. Our results are consistent with the ETS domain being an independently folded monomeric polypeptide with approximately 40% alpha-helix and a significant quantity of beta-sheet. The Elk-1 ETS domain is therefore of a mixed alpha/beta structure. Given the degree of conservation amongst other family members, it is likely that all ETS domains contain an alpha/beta DNA-binding motif. Structural predictions suggest that the ETS domain represents a novel structure with no obvious strong homology to any known DNA binding motifs(11, 12) . Several regions within the ETS domain are predicted to be alpha-helical(13, 62, 63) , which is consistent with our observation of the presence of significant amounts of alpha-helix. However, our detection of a significant proportion of beta-sheet within the ETS domain rules out the possibility that the ETS domain is predominantly alpha-helical as observed for the DNA-binding domains of the b-Zip (7) and bHLH proteins(4, 5, 6) . Moreover our results are consistent with the Elk-1 ETS domain being a correctly folded, single domain, polypeptide.

In summary, we have overexpressed, purified and characterized the ETS DNA-binding domain of Elk-1. By several functional and spectroscopic criteria, this domain is fully folded and functional. This work provides the framework around which further structural information about the ETS domain can be obtained. The determination of a high resolution three-dimensional structure for the ETS DNA-binding domain of Elk-1 should now be possible both in the presence and absence of its cognate binding site.


FOOTNOTES

*
This work was supported by the North of England Cancer Research Campaign and the Wellcome Trust. The Krebs Institute is a SERC Biomolecular Sciences Center for Molecular Recognition. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Holds a Biotechnology and Biological Sciences Research Council CASE studentship in association with Zeneca Pharmaceuticals.

To whom correspondence should be addressed. Tel.: 44-91-222-8800; Fax: 44-91-222-7424.

(^1)
The abbreviations used are: SRF, serum response factor; SRE, serum response element; bp, base pair(s); TOCSY, total correlated spectroscopy; NATA, N-acetyltryptophanamide.

(^2)
P. Shore and A. D. Sharrocks, unpublished data.

(^3)
A.D. Sharrocks and P. Shore, manuscript in preparation.

(^4)
L. Bisset, P. Shore, and A. D. Sharrocks, unpublished data.


ACKNOWLEDGEMENTS

We thank Peter Shaw for plasmid constructs, Bob Liddell for oligonucleotide synthesis, Margaret Bell for expert technical assistance and Susan Lee for secretarial assistance. We also thank Arthur Moir and Paul Brown for protein sequencing and helpful advice.

Note added in proof-While this manuscript was under review, two separate papers were published on the secondary structures of the ETS domains of Ets-1 (Donaldson, L. W., Peterson, J. M., Graves, B. J., and McIntosh, L. P.(1994) Biochemistry33, 13509-13516) and Fli-1 (Liang, H., Olejniczak, E. T., Mao, X., Nettesheim, D. G., Yu, L., Thompson, C. B., and Fesik, S. W.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 11655-11659). Our results on the ETS-domain of Elk-1 are supported by the conclusions in these two papers.


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