(Received for publication, April 4, 1997, and in revised form, May 2, 1997)
From the Department of Molecular Biology, Scripps
Research Institute, La Jolla, California 92037 and the ** Howard Hughes
Medical Institute, Salk Institute for Biological Studies,
La Jolla, California 92037
Unlike steroid and retinoid receptors, which associate with DNA as dimers, human estrogen related receptor-2 (hERR2) belongs to a growing subclass of nuclear hormone receptors that bind DNA with high affinity as monomers. A carboxyl-terminal extension (CTE) to the zinc-finger domain has been implicated to be responsible for determining the stoichiometry of binding by a nuclear receptor to its response element. To better understand the mechanism by which DNA specificity is achieved, the solution structure of the DNA-binding domain of hERR2 (residues 96-194) consisting of the two putative zinc fingers and the requisite 26-amino acid CTE was analyzed by multidimensional heteronuclear magnetic resonance spectroscopy. The highly conserved zinc-finger region (residues 103-168) has a fold similar to those reported for steroid and retinoid receptors, with two helices that originate from the carboxyl-terminal ends of the two zinc fingers and that pack together orthogonally, forming a hydrophobic core. The CTE element of hERR2 is unstructured and highly flexible, exhibiting nearly random coil chemical shifts, extreme sensitivity of the backbone amide protons to solvent presaturation, and reduced heteronuclear {1H-15N} nuclear Overhauser effect values. This is in contrast to the dimer-binding retinoid X and thyroid hormone receptors, where, in each case, a helix has been observed within the CTE. The implications of this property of the hERR2 CTE are discussed.
hERR1 1 and hERR2 are members of the
nuclear hormone receptor superfamily of transcription factors based
upon sequence homology (1, 2). They are derived from human kidney and
cardiac cDNA libraries, respectively (3). Members of this family
contain a highly conserved DNA-binding domain (DBD) of 66 amino acids, which consists of two zinc fingers, each of which coordinates a zinc
ion tetrahedrally with four cysteine thiolates. Much of what is known
mechanistically about this family has been derived from functional
(4-6) and structural (7-13) studies of the glucocorticoid, estrogen,
thyroid hormone, and retinoid receptors, which bind as dimers to direct
or inverted repeats of an AGGTCA or AGAACA response sequence (called
half-site). The hERR differs from these classical nuclear hormone
receptors in that (a) it has no known activating ligand,
making it an "orphan receptor"; and (b) the intact
protein as well as the isolated DBD bind as a monomer to a 5-extended
version of the estrogen/retinoid receptor half-site: TCAAGGTCA.2 Similar response
elements are recognized by a number of key monomer-binding orphan
receptors, including murine steroidogenic factor-1/embryonal long
terminal repeat-binding protein (14, 15), bovine Ad4-binding protein
(16), rat nerve growth factor I-B (17), and Drosophila FTZ-F1 (fushi tarazu
factor) (18). In particular, steroidogenic factor-1
recognizes CCAAGGTCA, is a major regulator of the expression of
steroidogenic enzymes (19), and has been implicated in sex
determination (20). hERR1 has recently been found to activate the
expression of human lactoferrin in endometrium and mammary gland cell
lines and hence must play a role in modulating immune responses and
cell growth (21).
Although there is currently no structural data available on
monomer-binding nuclear hormone receptors, several
functional/mutagenesis studies have identified a carboxyl-terminal
extension (CTE) of the zinc-finger modules as a second structural
determinant that contributes to DNA binding. Studies of the hERR
homolog, FTZ-F1, have identified a 30-residue CTE termed the FTZ-F1 box
(residues after Glu-171 in hERR2), which contributes a factor of 1000 to the binding constant; this region is proposed to be responsible for
binding the 5-end base pairs in the FTZ-F1 response element (22). In
contrast, a shorter 12-amino acid CTE in the RXR termed the T box is
helical and has been implicated in providing protein-protein interactions that stabilize the homodimer (12, 23). Furthermore, a
chimeric protein, generated between the zinc fingers of the RXR and a
carboxyl-terminal element in nerve growth factor I-B called the A box,
binds as a monomer to the nerve growth factor I-B response element
(23). Thus, while the CTE of the RXR is helical and contributes to the
binding affinity via protein-protein interactions, the monomer-binding
receptors have an as yet structurally uncharacterized CTE that provides
additional binding affinity via protein-DNA interactions. The present
NMR studies of the hERR2 DBD, which consists of the zinc-finger modules
and the CTE, represent a first step toward defining the structural
basis of these interactions.
The hERR2 DBD
(residues 96-194) was expressed in BL21 cells using the pGEX-2T vector
(Pharmacia Biotech Inc.), which produces an amino-terminal fusion
protein with glutathione S-transferase. Unlabeled protein
was expressed at 37 °C in Luria-Bertani broth by induction for
4 h with 0.5 mM
isopropyl--D-thiogalactopyranoside once cells had
reached A600 ~ 0.8. 15N-Labeled
protein was obtained using a minimal medium at pH 7.2 containing 44 mM Na2HPO4, 22 mM
KH2PO4, 9 mM NaCl, 1 mM
MgSO4, 0.1 µM CaCl2, 0.02 µM FeCl3, 0.1 mM
ZnCl2, 1% glucose, 1 mg/liter thiamine, and 1 g/liter
(15NH4)2SO4 (Cambridge
Isotope Laboratories). Cells were pelleted and resuspended in lysis
buffer containing 50 mM Tris (pH 8.0), 250 mM
KCl, 5 µM ZnCl2, 30 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100.
After one freeze-thaw cycle, cells were lysed by sonication. The high
DTT concentration was necessary to recover the hERR2 fusion protein
from the pellet since there was extensive protein cross-linking via
disulfide bonds. The sonicate was cleared by centrifugation for 15 min
at 15,000 × g. Glutathione-Sepharose 4B beads
(Pharmacia) were added to the crude cell lysate along with 100 mM DTT and 100 µM ZnCl2. After a
1-h incubation at 5 °C, the fusion protein·glutathione-Sepharose
complex was pelleted and washed three times with a phosphate-buffered
saline solution containing 100 mM DTT and 1 mM
ZnCl2. The first wash was done with 0.8 M NaCl
to remove DNA. The pelleted protein/beads were then resuspended in 50 mM Tris (pH 8.0), 150 mM NaCl, 2 mM
CaCl2, 100 mM DTT, and 100 µM
ZnCl2 containing 50 units/ml bovine thrombin (Sigma) and
incubated at room temperature until all of the hERR2 DBD had been
cleaved from the glutathione
S-transferase·glutathione-Sepharose complex, at which
point the proteolysis reaction was quenched with 1 mM
phenylmethylsulfonyl fluoride. The eluted protein was concentrated with
an Amicon flow cell (YM-3) and purified by HPLC using a C18
reverse-phase column (Vydac). After an initial wash with 27%
acetonitrile and 0.1% trifluoroacetic acid, protein was eluted with a
0.6%/min acetonitrile gradient. Fractions containing the hERR2 DBD
were lyophilized and weighed. The yield of pure hERR2 (>97%) was 2 mg/l of culture for labeled protein and 10-15 mg/liter for unlabeled
protein. Protein was homogeneous based upon SDS-polyacrylamide gel
electrophoresis, HPLC, and electrospray mass spectral analysis
(calculated Mr = 11,416; observed
Mr = 11,417). Mass spectral analysis of the
15N-labeled hERR2 DBD indicated >97% incorporation of
label. The electrospray mass spectrum of the native hERR2 DBD showed an
Mr of 11,547 as expected for hERR2 with two
bound zinc atoms.
To improve the level of
expression of the hERR2 DBD in Escherichia coli and the
stability of the protein in vitro, a C163A mutation that
removes a non-coordinating sulfhydryl group and a number of silent
mutations designed to favor the biased codon usage in E. coli were introduced into the gene for the hERR2
DBD.3 NdeI and BamHI
sites flanking the DBD gene were also incorporated via polymerase chain
reaction. The NdeI/BamHI hERR2 DBD fragment was
cloned into pET24a behind a T7/lacO promoter (24), and the expression plasmid was then introduced into a lysogenic E. coli strain (BL21(DE3)). Uniformly 15N-enriched
protein sample was prepared using a minimal medium similar to that
described above, except that the concentrations of glucose and
(15NH4)2SO4 were 3 and
2 g/liter, respectively. 13C,15N-Labeled
protein was obtained with a minimal medium in which [13C6]glucose (ISOTEC) was used. Gene
expression was induced by adding isopropyl--D-thiogalactopyranoside at a cell density of
A600 ~ 1.0-1.2. 1 h after
isopropyl-
-D-thiogalactopyranoside induction, rifampicin
was added to the culture to a final concentration of 100 µg/ml; the
cells were incubated for another 5 h at 37 °C. The protein was
isolated and purified to homogeneity (>95%) following a protocol
similar to that used for the wild-type hERR2 DBD, with the exclusion of
the glutathione-Sepharose chromatography step. The yield of purified
DBD improved to 10 mg/liter of minimal medium.
Gel-shift assays were carried out using
32P-end-labeled oligonucleotides that represent the
recognition site for hERR2, with two additional GC base pairs added on
either end for stability (5-GCTCAAGGTCACG-3
and the complementary
strand). Labeling and assays were performed using standard procedures
(25). The gel was 5% acrylamide (1:50 bisacrylamide) in 45 mM Tris borate. Incubations were carried out for 30 min at
25 °C in 6% glycerol, 1 mM DTT, 10 mM Tris
(pH 8.0), 50 mM KCl, and 0.5 mM
MgCl2, with DNA at <3.7 nM, hERR2 (wild-type
and C163A) varied from 23 µM to 23 nM, and
poly(dI·dC) at 0.1 µg/µl.
The lyophilized hERR2 DBD samples were resuspended in 6 M urea containing 0.5 mM ZnCl2 and 100 mM DTT and immediately passed over a PD-10 gel filtration column (Pharmacia) equilibrated with NMR buffer (20 mM potassium phosphate (pH 6.5-6.7), 100 mM KCl, 0.3 mM ZnCl2, and 5% D2O). After the addition of 10 mM DTT-d10, the sample was concentrated with a Centricon 3 membrane to give a final hERR2 DBD concentration of 0.8-1.5 mM. All solvents used for sample preparation were purged with argon gas. Since the protein precipitated at pH <6.5, all NMR experiments were carried out at pH 6.5-6.7.
NMR SpectroscopyAll NMR spectra were recorded on Bruker DMX750, AMX600, and AMX500 spectrometers at 300 K. The 1H carrier was set on H2O/HOD in all experiments and shifted to the center of the amide region during acquisition in the HSQC experiments. Phase-sensitive detection was achieved using either time proportional phase incrementation (26) or TPPI-States (27) quadrature detection. A 1H-1H NOESY spectrum (28) was recorded with a 100-ms mixing time, while jump and return NOESY spectra (29) were recorded with 200-, 100-, and 50-ms mixing times. A NOESY spectrum with an 80-ms mixing time was also recorded in D2O. The NOESY spectra included a Hahn-echo period at the end of the pulse sequence to improve the base line (30, 31). TOCSY spectra (32) were recorded with DIPSI-2 (decoupling in the presence of scalar interactions) (33) spin-lock periods of 30 and 50 ms. Double quantum filtered COSY (34) and double quantum (35, 36) experiments were performed according to standard methods. Two-dimensional homonuclear data were recorded with 128-320 scans, 512 t1 increments, and 8192 data points/increment and at 500-MHz proton carrier frequency. A spectral width of 12,500 Hz was used in the F2 dimension, while spectral widths of 6250-7000 Hz were used in the F1 dimension (12,500 Hz in the double quantum experiment). Digital oversampling by a factor of 2 was used to reduce base-line distortion (37). Two-dimensional {1H-15N} HSQC and NOESY-HSQC (100-ms mixing time) spectra were acquired using standard methods (38, 39). Three-dimensional 15N NOESY-HSQC (40, 41) data were recorded with mixing times of 100 and 150 ms, using 128 t1 (1H) increments, 32 t2 (15N) increments, and 16 free induction decays of 1024 data points/increment pair. Three-dimensional TOCSY-HSQC data were recorded in the same manner, with a spin-lock period of 40 ms. All multidimensional data were processed using either a modified version of the FTNMR software provided by Dr. Dennis Hare (Hare Research) or the Triad software package (Tripos Associates). Spectra were Fourier-transformed after applying a lorentzian-to-gaussian weighting function in the directly detected dimension and a shifted sine bell weighting function in the indirectly detected dimension(s). The water signal was removed from all spectra by applying a low pass filter to the time domain data (42).
Three-dimensional constant-time HNCA and HNCOCA spectra were collected
on the 13C,15N-labeled C163A hERR2 DBD
following established methods (43); pulsed-field gradients were
incorporated in these schemes for solvent suppression. These spectra
were recorded at 600 MHz with 32 t1 increments
(15N carrier at 117.75 ppm, spectral width of 1429 Hz), 64 t2 increments (13C- carrier at 54 ppm, spectral width of 4225 Hz), and 16 free induction decays of 1024 data points for the directly detected 1H resonances
(carrier at 8.2 ppm, spectral width of 5434 Hz). Heteronuclear
{1H-15N} steady-state NOEs for the C163A
hERR2 DBD were also measured at 600 MHz using two-dimensional HSQC
experiments (44). To minimize magnetization transfer from water to any
rapidly exchanging amide protons, a 6-s relaxation/irradiation delay
was employed between passes through the pulse sequence, and water was
suppressed with flip-back and gradient pulses.
The recombinant hERR2 DBD
(residues 96-194) exhibited a high affinity for the 5-extended
half-site sequence, 5
-TCAAGGTCA-3
, in gel-shift assays.2
Analysis of the holoprotein by pneumatically assisted electrospray spectrometry gave a molecular mass 130 ± 2 Da higher than that of
the apoprotein, indicative of two tightly bound zinc ions/polypeptide (data not shown). These data are consistent with the prediction that
hERR2 is a zinc-finger DNA-binding protein (3).
NMR studies were
carried out on the recombinant DBD peptide, which contains the putative
66-amino acid (ZnCys4)2-type zinc-finger domain
and the requisite 26-amino acid CTE for high affinity monomer binding
to the extended half-site sequence (22, 45). 87% of the backbone
protons, 53% of the side-chain protons, and 86% of the backbone
nitrogens have been assigned for Leu-102-Arg-174. Sequence-specific
assignments were made using the NOE-based sequential assignment
procedures (46) and are summarized in the two-dimensional {1H-15N} HSQC spectrum (Fig.
1). Sequential dNN connectivities
in two-dimensional NOESY and NOESY-HSQC spectra were used to connect
most of the residues, with the help of two-dimensional TOCSY and COSY
spectra to identify spin systems. The double quantum spectrum was
especially helpful in resolving ambiguities in the assignment of
side-chain protons and in assigning glycine CH protons. Assignment
ambiguities due to overlap could usually be resolved using the
three-dimensional NOESY-HSQC and TOCSY-HSQC spectra in conjunction with
the higher resolution two-dimensional
{1H-15N} HSQC spectrum. In cases where both
15N and 1H shifts were overlapped, a high
resolution two-dimensional {1H-15N} HSQC
spectrum obtained at lower temperature (292 K) proved useful. The
sequential and medium-range NOE connectivities, summarized in Fig.
2, clearly identify two helices, but show no sign of
helix formation after the Gly-Met termination site for helix-2. Of the 77 backbone amide cross-peaks observed, 67 could be assigned to 68 residues, while the remainder exhibited no sequential connectivities. The majority of the unassigned residues are in what is a highly basic
loop region in finger-2 (residues 149-155) of other structurally characterized nuclear hormone receptors and in the carboxyl-terminal region. Resonances for 15 of the 20 residues C-terminal of Arg-174 could not be assigned since sequential and medium-range NOEs are either
weak or absent.
Secondary Structural Elements and Global Fold of the Wild-type hERR2 DBD
The medium-range NOEs summarized in Fig. 2 show one
13-residue helix (Glu-121-Gly-133) that starts at the
carboxyl-terminal end of finger-1 and a second 12-amino acid helix
(Gln-156-Gly-167) that starts at the carboxyl-terminal end of
finger-2. The region after helix-2 shows no sign of secondary structure
and may be disordered (see below). There is also a short antiparallel
-sheet between Cys-103/Leu-104 and Ser-119/Cys-120 based upon strong d
N connectivities and long-range NOEs between
Cys-103 C
H and Cys-120 NH and between Ser-119 C
H and Leu-104 NH.
This same
-sheet is also present in homologous sequences of the
retinoid X (12), retinoic acid (11), and glucocorticoid (7)
receptors.
It is also interesting to note that the Val-105 and Cys-106 amide
protons are two of the most persistent (along with three others
discussed below) in D2O (~30 h), suggesting that they are both protected from solvent and probably involved in strong internal hydrogen bonds. The equivalent residues in other zinc-finger proteins have been shown, through structural and/or
H2O/D2O exchange data, to be hydrogen-bonded to
S- of the first zinc-finger cysteine ligand (7, 12, 47-53). This
amide to S-
hydrogen bond may be present in hERR2 based upon
homology and the slow hydrogen/deuterium exchange rate.
On the basis of long-range NOEs that had been identified (Fig.
3A), the 66-amino acid zinc-finger domain
(i.e. up to the Gly-Met termination signal for helix-2) of
hERR2 consists of two helices that are packed against each other to
enclose a hydrophobic core consisting of Phe-126, Phe-127, Thr-130,
Ile-131, Phe-160, and Leu-164 (Fig. 3B). Long-range NOEs
between residues in or near this hydrophobic core and Val-173 indicate
that the region just past helix-2 also folds onto this hydrophobic
core. A similar overall pattern of long-range NOEs was observed in the
RXR (12), suggesting that this part of the structure has a fold very
similar to that of the retinoid X, estrogen (9, 10), glucocorticoid (7,
8), and retinoic acid (11) receptors. The slow exchange rates for the
amide protons of Cys-123, Lys-124, and Ala-125 suggest that these
residues may be buried in the protein interior, which is reasonable
since they reside on the amino-terminal end of helix-1, near the
hydrophobic core.
Role of Cys-163
Alignment of the sequence with those of structurally characterized receptor molecules suggests that Cys-163 of hERR2 is not involved in coordinating zinc ions. The homologous cysteines in these hormone receptors are found close to the hydrophobic core and are partially solvent-accessible. Assuming that Cys-163 has similar spatial orientation, it could facilitate aggregation via disulfide formation at the high protein concentrations and temperatures employed for the NMR experiments and cause sample degradation over extended periods of time. Hence, to increase the intrinsic stability and solubility of hERR2, Cys-163 was replaced with an alanine. Alanine was chosen because it has comparable hydrophobic character to cysteine since it is probable that Cys-163 is partly buried.
Gel-shift studies indicate that both the wild-type and C163A hERR2 DBDs
have comparable affinities for the extended half-site sequence (data
not shown). With the aid of the existing data for the wild-type hERR2
DBD, the proton and nitrogen chemical shifts were reassigned for the
mutant protein from two- and three-dimensional 15N-edited
spectra. Based on perturbations of amide proton and nitrogen chemical
shifts (Fig. 4), the mutation has little effect on the structure, other than minor perturbations of groups near the
hydrophobic core where Cys-163 is located. The perturbations of
15N shifts for some residues in finger-1 (Fig.
4A) are consistent with long-range NOE data (Fig.
3A) that indicate that this region of finger-1 participates
in the formation of the hydrophobic core.
The CTE Is Unstructured
It should be noted that the
three-dimensional 15N-edited spectra for the mutant protein
did not enable us to extend the backbone resonance assignments beyond
what was defined for the wild-type protein. Because of dramatically
improved expression levels with the mutant gene construct, a uniformly
13C,15N-labeled sample of the C163A hERR2 DBD
was prepared, and triple-resonance experiments were recorded. The
combination of three-dimensional HNCA and HNCOCA spectra allowed us to
establish inter-residue connectivities using 13C-
chemical shift values and to complete the backbone assignments for the
CTE of hERR2. The 13C-
chemical shift provides a
reliable index of the local secondary structure; a downfield or upfield
shift in
(13C-
) is indicative of a helix or
-stranded configuration, respectively (54, 55). Comparison of the
C-
chemical shifts in the mutant protein with random coil values
(Fig. 5A) reveals the same two helical
elements observed in the wild-type DBD. Furthermore, the analysis
establishes that the CTE does not adopt a regular secondary structure,
but has chemical shifts characteristic of a random coil. The absence of
long-range NOEs and most sequential NOEs for residues C-terminal of
Arg-174 also provides strong evidence that the CTE is unstructured.
It has been generally surmised that unstructured regions of a protein are characterized by increased main-chain flexibility. The dynamic properties of the DNA-binding domain of hERR2 in solution were evaluated by measuring heteronuclear {1H-15N} NOEs for the C163A variant. The magnitude of the steady-state {1H-15N} NOE is highly sensitive to the dynamics of the 1H-15N vector; negative or small positive NOE values are diagnostic of significantly fast motions relative to the overall tumbling rate of the molecule. Residues in the CTE and the N terminus of the C163A hERR2 DBD exhibit diminished heteronuclear NOEs relative to those in the zinc-finger core (Fig. 5B). The loop (Thr-148-Ser-155) between the sixth and seventh ligating cysteines represents the most flexible region of the zinc-finger core. In the CTE, the heteronuclear NOE begins to decrease C-terminal of Val-173, with near zero values reported for Arg-179-Arg-187 and negative values observed for the last six residues. This trend in heteronuclear NOE for the CTE is consistent with an unstructured polypeptide tethered to the folded zinc-finger domain. The flexible nature of the CTE also explains the absence of sequential and long-range interactions involving this region that could be identified from the two- and three-dimensional NOESY data sets. The evidence indeed supports that the functionally critical CTE of hERR2 is dynamically disordered.
The region encompassing residues 103-168 of hERR2 shares a strong
sequence homology with the zinc-finger domains of numerous members of
the nuclear hormone receptor superfamily. This domain and a region
(CTE) immediately downstream of it comprise the minimum motif for
site-specific 1:1 binding to an extended version of the canonical
half-site sequence (5-TCAAGGTCA-3
). We have constructed a recombinant
form of the DBD of hERR2 and shown that it binds two zinc(II)
ions/polypeptide. NMR structural analysis of the DBD indicates a
polypeptide fold consisting of two orthogonally packed helices within
the putative zinc-finger domain; this fold is typical of those observed
in steroid and retinoid receptors. These data confirm that hERR2 is a
member of the nuclear hormone receptor family of zinc-finger
transcription factors.
The zinc-finger domains of nuclear hormone receptors contain nine highly conserved cysteines, yet only the eight N-terminal cysteines are involved in coordinating the zinc ions. We have replaced the ninth cysteine (Cys-163) of hERR2 with an alanine. Comparison of the functional and solution NMR properties of the mutant with those of the wild-type protein suggests that Cys-163 is important neither for the proper folding of the polypeptide around the zinc sites nor for binding of the DBD to the cognate DNA. We cannot, however, exclude a functional role for this invariant cysteine in vivo.
It is the 26-amino acid CTE that is of greatest functional interest in
hERR2 since the equivalent regions in nerve growth factor I-B (23),
steroidogenic factor-1 (19), and FTZ-F1 (FTZ-F1 box) (22) have been
shown to provide key interactions for monomeric instead of dimeric
binding to DNA (see above). The region has further been proposed to
provide sequence-specific contacts with the 5-base pairs in
the response element (TCAAGGTCA in hERR2) (45). Prior to
this work, no structural information was available on the CTE elements
of monomer-binding receptors. The CTE of the hERR2 DBD (Fig.
3A) contains sequences corresponding to the T and A boxes in
a number of structurally characterized dimer-binding receptors. In the
RXR, the T box is helical and has been implicated in stabilizing the
homodimer via protein-protein interactions (12). In the ternary complex
of the thyroid hormone receptor, the RXR, and DNA (13), the CTE of the
thyroid hormone receptor does not participate in the dimer interface.
Instead, the T box serves as a connecting loop to a long helical A box,
which makes extensive nonspecific contacts with the DNA
backbone. It is evident that the CTE of the thyroid hormone receptor
has a different functional role than those of the monomer binders.
Unlike those of the RXR and the thyroid hormone receptor wherein
helices have been observed, the carboxyl-terminal T/A box region of
hERR2 appears to be disordered, having no identifiable secondary
structure. This is based on (a) C- chemical shifts (Fig.
5A) that approach random coil values; (b) reduced
heteronuclear {1H-15N} NOEs (Fig.
5B); (c) the appearance of an additional six HSQC peaks when gradient pulses or spin-lock purge pulses are used, rather
than presaturation, for water suppression (data not shown); and
(d) sequential NOEs that are either weak or absent. Since the CTE contributes to both affinity and specificity, it may adopt a
defined secondary structure upon DNA binding as in the trp
repressor or glucocorticoid receptor (8, 56), or it may adopt an
ordered but extended structure as in GATA-1 (57). Preliminary NMR
studies of the C163A hERR2-DNA complex suggest that ordering of the CTE does occur upon binding to DNA,3 and studies of the
structure of the complex are currently in progress.
Supplementary tables are available at the BioMagRes Bank at the University of Wisconsin, BMRB accession numbers 4033 (wild type) and 4034 (C163A).
We thank Min Lee, John Chung, Signe Holmbeck, and John Cavanagh for technical assistance and stimulating discussions.