extfieldsofstudy: Chemistry Medicine

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The specific interaction of dinitrosyl-diglutathionyl-iron complex, a natural NO carrier, with the glutathione transferase superfamily: suggestion for an evolutionary pressure in the direction of the storage of nitric oxide.

The interaction of dinitrosyl-diglutathionyl-iron complex (DNDGIC), a natural carrier of nitric oxide, with representative members of the human glutathione transferase (GST) superfamily, i.e. GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2, has been investigated by means of pre-steady and steady state kinetics, fluorometry, electron paramagnetic resonance, and radiometric experiments. This complex binds with extraordinary affinity to the active site of all these dimeric enzymes; GSTA1-1 shows the strongest interaction (KD congruent with 10-10 m), whereas GSTM2-2 and GSTP1-1 display similar and slightly lower affinities (KD congruent with 10-9 m). Binding of the complex to GSTA1-1 triggers structural intersubunit communication, which lowers the affinity for DNDGIC in the vacant subunit and also causes a drastic loss of enzyme activity. Negative cooperativity is also found in GSTM2-2 and GSTP1-1, but it does not affect the catalytic competence of the second subunit. Stopped-flow and fluorescence data fit well to a common minimal binding mechanism, which includes an initial interaction with GSH and a slower bimolecular interaction of DNDGIC with one high and one low affinity binding site. Interestingly, the Theta class GSTT2-2, close to the ancestral precursor of GSTs, shows very slow binding kinetics and hundred times lowered affinity (KD congruent with 10-7 m), whereas the bacterial GSTB1-1 is not inhibited by DNDGIC. Molecular modeling and EPR data reveal structural details that may explain the observed kinetic data. The optimized interaction with this NO carrier, developed in the more recently evolved GSTs, may be related to the acquired capacity to utilize NO as a signal messenger.

Glutathione S-transferases (GSTs) 1 (EC 2.5.1.18) are a superfamily of multifunctional enzymes able to protect the cell against endogenous or exogenous toxic compounds (1). The human cytosolic GSTs are homo-or heterodimeric proteins, encoded by distantly related gene families and grouped into eight classes termed Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta (2)(3)(4)(5)(6)(7)(8) on the basis of sequence similarity, substrate and inhibitor specificity, immunological properties, and three-dimensional structure. The most important reaction catalyzed by GSTs is the conjugation of the sulfur atom of GSH to an electrophilic center of many toxic organic compounds. The Alpha class also shows an additional selenium-independent peroxidase activity with organic peroxides (9). Other physiological roles of GSTs include chemical sequestration (10), regulation of the Jun kinase protein (11), inhibition of the proapoptotic action of Bax (12), and modulation of calcium channels which oppose the apoptotic mobilization of calcium ions (13). In the last 10 years, the x-ray structures of representative members of the GST superfamily have been solved, showing similar three-dimensional folding of these proteins and very similar topography of the GSH-binding sites (G-sites) (14 -17). Recently, we described the peculiar interaction between GSTP1-1 and dinitrosyl-diglutathionyl-iron complex (DNDGIC) (Scheme 1), a natural nitric oxide carrier that binds to the G-site with thousand times higher affinity than GSH (18). DNDGIC, which can be formed in vivo by free NO or NO donors, GSH, and traces of ferrous ion, is a paramagnetic molecule with a diagnostic electron paramagnetic resonance (EPR) spectrum centered at about g ϭ 2.03 both in the frozen state at 77 and at 298 K (19). The occurrence of dinitrosyl-iron complexes (DNICs) in mammalian tissues and cells was observed more than 30 years ago after exposure of tissues to endogenous or exogenous NO (19). In vivo, DNDGIC and other low mass DNICs could be in equilibrium with several protein-bound forms after replacing one or both the free thiol ligands with protein residues like His, Cys and Ser (19). Both low mass and high mass DNICs seem to be more stable than NO and may possibly act as storage of nitric oxide (20 -22) as well as intermediates in the iron-catalyzed formation and decomposition of S-nitrosothiols (23). DNICs also inhibit platelet aggregation (24), reduce blood pressure (25), relax vascular vessels (26), induce accumulation of heat shock protein HSP70 (27,28), and modulate ion channel activity (29). The recently discovered interaction of DNDGIC with GSTP1-1 is of particular interest as this complex possibly represents the most potent natural competitive inhibitor of this enzyme (18). A particular intersubunit communication triggered by DNDGIC was also found, which lowers the affinity of the adjacent subunit. EPR spectroscopy and molecular modeling indicated that DNDGIC is stabilized in the G-site through the usual polar and hydrophobic interactions of protein resi-dues with one GSH molecule, coordination of the iron ion to the hydroxyl group of Tyr-7, and additional van der Waals interactions of NO moieties with Ile-104 and Tyr-108 (18). This interaction is quite surprising as it is a common opinion that the G-site recognizes GSH (and a few GSH analogs) almost exclusively. In addition, cooperative intersubunit communication in GSTP1-1 has only been observed after a few selected point mutations (30,31) or under thermal stress (32) but never in the native enzyme under physiological conditions. The tight interaction between GSTP1-1 and an endogenous NO carrier is also indicative of an additional physiological role for this enzyme as an NO-storage protein. The surprising results of this paper demonstrate that not only GSTP1-1 but all representative members of mammalian GSTs interact with DNDGIC showing similar binding mechanism and cooperativity.

EXPERIMENTAL PROCEDURES
Enzyme Expression and Purification-Human GSTA1-1, GSTM2-2, and GSTP1-1 were expressed in Escherichia coli and purified as described previously (18,33,34). Expression of the human GSTT2-2 and its purification using immobilized metal ion chromatography on a nickel-nitrilotriacetic acid matrix (Qiagen, Hilden, Germany) were carried out as reported previously (35,36). Expression and purification of the bacterial GSTB1-1 was performed as described in Ref. 37. The enzyme concentrations reported in the text for all GSTs refer to the single subunit.
DNDGIC Synthesis-DNDGIC was prepared essentially as described previously (18). Briefly, 1 ml of 0.5 mM FeSO 4 (dissolved in degassed water to avoid rapid oxidation to the ferric state) was added to 10 ml (final volume) of 0.1 M of potassium phosphate buffer, pH 7.4, containing 20 mM GSH and 2 mM GSNO (25°C). After 10 min, the reaction was almost complete, and the resulting stock solution of DNDGIC (50 M) was stable for at least 3 h. For the sake of simplicity, we have used the abbreviation DNDGIC also for the complex bound to GSTs, although the latter is shown to be a monoglutathionyl species.
Inhibition Experiments-Inhibition of GSTA1-1, GSTP1-1, and GSTM2-2 by DNDGIC was studied by incubating variable amounts of DNDGIC (from 0.4 to 9 M) with a fixed enzyme concentration (4 M) in 1 ml of 0.1 M potassium phosphate buffer, pH 7.4 (25°C). After 2 min, 4-l aliquots were diluted in 1 ml of 0.1 M potassium phosphate buffer, pH 6.5, containing 10 mM GSH and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme activity was determined spectrophotometrically at 340 nm (25°C) (⑀ ϭ 9,600 M Ϫ1 cm Ϫ1 for the DNB-GSH adduct) within 10 s from addition of the substrates. This procedure, mentioned in the text as "assay under non-equilibrium conditions" gives a snapshot of the binding situation as it occurs in the incubation mixture and not in the final assay solution because of the slow extrusion of DNDGIC by 10 mM GSH (see "Results"). The inhibition of the low affinity binding site was followed by incubating variable amounts of DNDGIC (from 0.1 to 10 M) with 40 nM of GSTs (20 nM for GSTM2-2) in 1 ml of 0.1 M potassium phosphate buffer, pH 7.4. After 5 min, the pH was adjusted to 6.5, and the concentration of GSH was brought to 10 mM, and 1 mM of CDNB was added rapidly. Residual activity was determined within 10 s from addition of substrates. GSTT2-2 (1.5 M) was incubated at 37°C with variable amounts of DNDGIC (from 0.75 to 15 M) in 1 ml of 0.1 M potassium phosphate buffer, pH 7.4. After 10 min, 10 mM GSH and 0.25 mM menaphthyl sulfate (Msu) were added for activity measurements at 298 nm (38). GSTB1-1 (1 M) was incubated at 25°C with variable amounts of DNDGIC (from 0.75 to 15 M) in 1 ml of 0.1 M potassium phosphate buffer, pH 7.4. After 10 min, the pH was adjusted to 6.5, and 10 mM GSH and 1 mM CDNB were rapidly added for activity measurements at 340 nm.
Inhibition experiments termed "under equilibrium conditions" were performed as described above with the exception that DNDGIC was incubated with each enzyme for 5 min (40 min for GSTT2-2) in the presence of a fixed GSH concentration (10 mM). After incubation with DNDGIC, aliquots were diluted in the assay mixture containing 10 mM GSH. After 5 min (40 min for GSTT2-2), 1 mM CDNB (1 mM menaphthyl sulfate for GSTT2-2) was added for activity determination. Data were fitted to a bi-exponential decay equation.
Fluorescence Experiments-Quenching of intrinsic fluorescence by DNDGIC was measured in a single photon counting spectrofluorometer (Fluoromax, S.A. Instruments, Paris, France) with a sample holder thermostatted at 25°C (37°C for GSTT2-2). Excitation was at 280 nm and emission at 340 nm. In a typical experiment GST (2 M) was incubated with variable amounts of DNDGIC (from 0.2 to 20 M) in 1 ml of 0.1 M potassium phosphate buffer, pH 7.4, containing 10 mM GSH and 1 mM GSNO. After 5 min (40 min for GSTT2-2), the fluorescence at 340 nm was measured and corrected for inner filter effect. Data were fitted to a bi-exponential decay equation.
Pre-steady State Kinetic Experiments-Rapid kinetic experiments were performed on a Applied Photophysics Kinetic spectrometer stopped-flow instrument equipped with a 1-cm light path observation chamber thermostatted at 25°C (37°C for GSTT2-2). In a typical experiment 4 M GST in 0.1 M potassium phosphate buffer, pH 7.4, was rapidly mixed with an identical volume of the same buffer containing 5 M DNDGIC, 20 mM GSH, and 2 mM GSNO. The reaction was followed by fluorescence changes at 320 nm (excitation at 280 nm; bandwidth 20 nm) and repeated at three different complex concentrations (5, 15, and 45 M) and four different GSH (from 2 to 60 mM) and GSNO (from 0.2 to 6 mM) concentrations keeping the ratio GSH:GSNO constant at 10. Thousand data points were obtained on a logarithmic time scale. The experimental traces obtained at nine different DNDGIC and GSH (plus GSNO) concentrations were simultaneously fitted to Scheme 2 using the program Gepasi 3.21 (39 -41). The program carries out the nonlinear minimization of the kinetic constants by means of numerical integration, at variable steps, of the ordinary differential equation according to Scheme 2, where E is each subunit with high affinity, E* is the subunit with low affinity, C is DNDGIC, and E⅐GSH is the pre-complex found in the GSH binding mechanism to GSTA1-1, GSTP1-1, and GSTM2-2 (42). GSH binding to GSTT2-2 lacks the pre-complex intermediate (43).
EPR Experiments-Samples for EPR experiments were usually prepared in 0.1 M potassium phosphate buffer, pH 7.4, with DNDGIC added from a freshly made stock solution. EPR measurements were made at room temperature (22-25°C) with a Bruker ESP300 X-band instrument (Bruker, Karlsruhe, Germany) equipped with a high sensitivity TM 110 -mode cavity. To optimize instrument sensitivity, spectra were recorded using samples of 80 l contained in flat glass capillaries (inner cross-section 5 ϫ 0.3 mm) (44). For kinetics measurements standard round glass capillaries (1.10 mm inner diameter) with a working volume of 40 l were applied, to allow rapid handling of samples. Unless otherwise stated spectra were measured over a 200 G range using 20 milliwatts power, 2.0 G modulation, and a scan time of 42 s; typically 4 -40 single scans were accumulated to improve the signal to noise ratio. High resolution spectra were recorded with 0.1 G modulation and 2 milliwatts power. Simulation of anisotropic spectra was carried out using the SimFonia software provided by Bruker.
Radiometric Dinitrosyl-Diglutathionyl-Iron Complex and GST Superfamily site (more than 90%) was estimated on the basis of the observed inhibition. The solution was rapidly filtered on a Whitman 3MM filter paper. The filter was washed twice with 1 ml of cold buffer to remove excess of [ 3 H]GSH and of unbound [ 3 H]DNDGIC. The dried filter was dipped into 3 ml of Optifluor and, after 6 h, the radioactivity was determined by liquid scintillation spectrometry. Blank experiments, in the absence of FeSO 4 or GSTA1-1 were performed to evaluate the amount of spurious radioactivity retained by the filter or by the protein.
DNDGIC Exchange between GSTM2-2 and GSTA1-1-GSTM2-2 (2.5 M) was reacted for 5 min with 1.25 M DNDGIC in 0.1 M potassium phosphate buffer, pH 7.4. Residual activity determined with CDNB was 52% of the initial value. The enzyme was then added to an identical volume of the same buffer containing 2.5 M GSTA1-1. By taking advantage of the different specific activities of GSTA1-1 and GSTM2-2 toward CDNB (80 and 200 units/mg, respectively) and NBD-Cl (40 and 3 units/mg, respectively), the concentration of the bound and free forms of each enzyme was calculated at fixed incubation times.
Molecular Modeling-Molecular modeling was performed by using the program Insight II (release 2000; MSI, San Diego). The models were based on the crystallographic coordinates of the different GSTs in complex with GSH, as obtained from the Brookhaven Protein Data Bank: A1-1, entry 1guh (2.6 Å resolution); M2-2, entry 1hna (1.85 Å resolution); P1-1, entry 6gss (1.9 Å resolution); and T2-2, entry 1ljr (3.2 Å resolution). The procedure followed was very similar to that already reported for GST P1-1 (18); the GSH-dinitrosyl-iron complex model was docked in the active site of the enzyme by assuming that the GSH molecule was in the same position and conformation as in the crystal structure of the enzyme-GSH complex. After adding the iron atom and the two NO molecules by assuming a tetrahedral geometry for the complex, it was immediately evident that the hydroxyl moiety of a Tyr or Ser residue (Tyr-9 in GST A1-1, Tyr6 in M2-2, Tyr-7 in P1-1, and Ser-11 in T2-2) was always at the appropriate distance and orientation to act as the fourth ligand of the iron atom, and therefore this coordination bond was added. Bond distances and angles were determined by the Builder module of the Insight II program. Finally the model was energy-minimized to convergence while keeping all the protein atoms fixed (except those of the Tyr or Ser residue), using the Discover module of Insight II and the CVFF force field (45). For comparison purposes, a similar minimization was performed also for the GSH molecule in the crystallographic enzyme-GSH complex. Graphic representation of the final model was produced by the program MOLMOL (46).

RESULTS
Inhibition Experiments-In a first experimental approach, the interaction of DNDGIC with representative members of the GST superfamily was studied on the basis of the competitive inhibition by DNDGIC. Under the experimental conditions used previously for GSTP1-1 (18), the inhibition of GSTM2-2 at variable DNDGIC concentrations shows a biphasic behavior close to that found for GSTP1-1, suggesting a similar cooperative mechanism that lowers the affinity of the vacant subunit for DNDGIC ( Fig. 1). Binding to the high affinity G-site is so efficient that low amounts of DNDGIC inhibit stoichiometrically up to about 50% of the original activity. Because it is not possible to determine the trace amounts of free complex in solution in this first phase, only an apparent upper limit value of K i1 Յ 10 Ϫ11 M can be estimated for the high affinity binding site, whereas the affinity of the low affinity binding site can be calculated more accurately (K i2 ϭ 1.3 ϫ 10 Ϫ8 M). The cooperative mechanism triggered by DNDGIC binding does not modify the affinity for GSH as suggested by an unchanged K m value found in halfinactivated GSTM2-2 (data not shown). The Alpha class GSTA1-1 also shows strong affinity (apparent K i1 Յ 10 Ϫ11 M), but only 10% of the total activity remains when one active site of the dimeric protein binds one DNDGIC (Fig. 1). Because of the very low residual activity of the second subunit, only an approximate K i2 value of about 10 Ϫ8 M can be evaluated for the low affinity binding site. The inhibition of the vacant active site is not caused by a change of affinity for GSH. In fact, the half-complexed enzyme with DNDGIC shows an unchanged K m value for GSH (0.18 mM). A negative influence of DNDGIC bound to one subunit on the deproto-nation of GSH (crucial in catalysis) in the adjacent subunit is also unlikely. In fact, the k cat versus pH plot overlaps that found for the native enzyme, showing that the pK a value of GSH is unchanged (experiments not shown). Thus, unproductive binding of GSH is possibly involved in the observed overinhibition. GSTT2-2 shows a different behavior. This enzyme, probably developed before Alpha, Pi, and Mu class GSTs in the evolutionary pathway (43,47), shows a small and buried G-site, and a Ser residue (Ser-11) replaces the Tyr residue found in the more recently evolved GSTs which is essential for the activation of the bound GSH. GSTT2-2 displays a hundred times lower affinity for DNDGIC (apparent K i1 Յ 4 ϫ 10 Ϫ6 M, Fig. 1), whereas the biphasic inhibition pattern suggests a similar negative cooperativity. The high concentration of enzyme used in these kinetic experiments (due to the very low specific activity of GSTT2-2) and the low concentration of the DNDGIC stock solution do not allow measurement of reliable K i2 values for the low affinity binding site. Interestingly, the bacterial GSTB1-1, which binds GSH with low affinity and lacks a Tyr or Ser residue for GSH . After 2 min, the pH was adjusted to pH 6.5; the GSH concentration was brought to a final concentration of 10 mM, and 1 mM CDNB was then added for activity measurement. c, GSTT2-2 ࡗ (1.5 M) and GSTB1-1 Ⅺ (1 M) were incubated at pH 7.4 (25°C for GSTB1-1 and 37°C for GSTT2-2) with variable amounts of DNDGIC (from 0.75 M to 15 M). After 10 min the pH was adjusted to 6.5 for GSTB1-1, and activity measurements were done by adding 10 mM GSH and 1 mM CDNB (25°C). Activity measurements for GSTT2-2 were performed at pH 7.4 (37°C) after addition of 10 mM GSH and 0.25 mM Msu. Activity is expressed as percentage of the original value. activation, shows an unchanged activity even in the presence of 10 moles excesses of DNDGIC ( Fig. 1), indicating that this more primitive enzyme has scarce affinity for this complex.
Apparently all inhibition experiments reported above gave reliable data, but caution must be taken in kinetic studies involving tight binding inhibitors. It is not unusual that they need minutes and sometimes hours to reach steady state conditions. Thus, further analysis is required to check possible occurrence of slow reacting species in the DNDGIC-GST system.
DNDGIC Binding and Extrusion from the Active Site Are Slow Events-Binding of stoichiometric amounts of DNDGIC to the G-site of all representative GST members is a relatively slow event. As expected on the basis of a reciprocal competition of GSH and DNDGIC for the same G-site, the presence of increasing concentrations of GSH lowers the rate of the binding process and the extent of the final inhibition. By using 1 mM GSH, 2 M enzyme, and 2 M DNDGIC, kinetics of binding has t1 ⁄2 values of 7, 13, and 11 s for GSTA1-1, GSTM2-2, and GSTP1-1, respectively (Fig. 2). GSTT2-2 exhibits the lowest rate of DNDGIC binding with a t1 ⁄2 of 100 s (Fig. 2). In the presence of 10 mM GSH (the highest GSH concentration present in our kinetics experiments), a reasonable equilibrium (i.e. constant inhibition) is reached only after 5 min of incubation for Alpha, Pi, and Mu GSTs (25°C), whereas 40 min are required for GSTT2-2 at 37°C (data not shown). Displacement of DNDGIC by GSH is also a slow process. When the enzyme is first reacted with DNDGIC in 1 mM GSH and then incubated with 10 mM GSH, a time-dependent partial reactivation occurs, characterized by t1 ⁄2 values of 40 s for GSTA1-1 and GSTP1-1, 80 s for GSTM2-2, and 1000 s for GSTT2-2 (data not shown). Kinetics of reactivation does not change by varying the final GSH concentration from 5 to 20 mM, and this means that GSH binding is rate-limited by the release of the complex from the G-site. In other words, apparent k reactivation values may be assumed to correspond to the apparent k off values of the competitive inhibitor. These kinetic findings suggested conditions to be used for inhibition experiments under equilibrium conditions (see "Experimental Procedures"). Under these new assay conditions, negative cooperativity is still evident for GSTM2-2, GSTP1-1, and GSTT2-2, and more accurate K i values can be calculated ( Fig. 3 and Table I) in the presence of constant 10 mM GSH concentration. GSTA1-1 displays the highest affinity (K i1 ϭ 8.0 ϫ 10 Ϫ11 ), and a similar K i1 value of about 1.0 ϫ 10 Ϫ9 M has been found for the high affinity binding site of Pi and Mu GSTs (8.0 ϫ 10 Ϫ8 and 2.0 ϫ 10 Ϫ8 M for their low affinity binding sites, respectively). The Theta GSTT2-2 shows the lowest affinity with K i1 ϭ 3.0 ϫ 10 Ϫ7 M and K i2 ϭ 4.0 ϫ 10 Ϫ6 M.
We must emphasize that the kinetic data obtained under "non-equilibrium" conditions, i.e. without a preincubation step of the inhibited enzyme with 10 mM GSH in the assay mixture, are of particular interest. Because of the slow reactivation kinetics by 10 mM GSH, they provide a snapshot of the DNDGIC-GST interaction as it occurs immediately after exposure to the complex and not in the final assay mixture. This allows us to evaluate the binding stoichiometry (see Fig. 1) involving the high affinity G-site and to check the interaction of DNDGIC with GSTs in heterogeneous biological systems as described in the accompanying paper (48).
FIG. 3. Inhibition of different GST isoforms by DNDGIC under equilibrium conditions. GSTA1-1, GSTM2-2, and GSTP1-1 were incubated for 5 min with variable amounts of DNDGIC at pH 7.4 in the presence of 10 mM GSH as described under "Experimental Procedures." Aliquots were then diluted in the assay mixture containing 10 mM GSH. After 5 min of incubation, 1 mM CDNB was added for activity determination. The final enzyme concentration in the assay was 40 nM (20 nM for GSTM2-2). GSTT2-2 (1.5 M) was incubated at pH 7.4 and 37°C with variable amounts of DNDGIC (from 3.7 to 37 M) and constant GSH (10 mM). After 40 min 0.25 mM Msu was added for activity assay. Continuous lines are the best fit of the experimental points obtained for GSTP1-1, GSTM2-2, and GSTT2-2 to a bi-exponential decay equation. The continuous line for GSTA1-1 is the best fit to a monoexponential decay equation. K i1 and K i2 represent the inhibition constants for the high and low affinity binding sites. Fluorescence Experiments-Binding of DNDGIC to GSTs causes a significant perturbation of the intrinsic fluorescence at 340 nm (Fig. 4), useful to estimate directly the thermodynamic dissociation constant of the DNDGIC-enzyme complex in the absence of CDNB as co-substrate. Fluorescence experiments have been carried out at fixed GSH and GSNO concentrations (10 and 1 mM, respectively), and the fluorescence quenching by DNDGIC was corrected for the fluorescence perturbation due to GSH and GSNO. Data reported in Fig. 4 fit well to a binding equation that includes one high affinity and one low affinity binding site (see Table I). Because of the relatively high concentrations of enzyme and complex used in these experiments, an almost stoichiometric binding of the inhibitor to the high affinity G-site has been found, so only an upper limit of the K D value can be estimated. Interestingly, the fluorescence approach allowed us to visualize well the binding of DNDGIC to the low affinity G-site of GSTA1-1. This event could not be characterized accurately by inhibition experiments as the hemisaturated enzyme is almost inactive (see Fig. 1).
Stopped-flow Experiments-A more detailed and conclusive kinetic investigation on the binding of DNDGIC to the representative members of the GST superfamily has been done by stopped-flow experiments, by following the quenching of intrin-sic fluorescence at 340 nm after rapid addition of DNDGIC to each enzyme (Fig. 5). The experimental traces obtained with a set of experiments, performed by rapid mixing of GSTA1-1, GSTM2-2, and GSTP1-1 with variable amounts of DNDGIC at constant GSH and GSNO concentrations, show two distinct phases. A first very fast fluorescence quenching due to the binding of GSH (or GSNO) to the G-site is followed by a slower fluorescence quenching attributed to the binding of the complex. Most of the first fast interaction of the enzyme with GSH is lost in the instrumental dead time and can be followed adequately only at 5°C, thus being characterized by the same microscopic rate constants reported previously for the GSH binding (42,43). Thus, the presence of DNDGIC (several orders of magnitude less concentrated than GSH) and GSNO (10 times less concentrated than GSH) seems to have no relevant effect on this first event, i.e. GSH binding. The second slower phase, i.e. DNDGIC displacing GSH, occurs on a very different time scale (Fig. 5). All experimental traces for GSTA1-1 and GSTP1-1 have been fitted well to a minimal binding mechanism (see Scheme 2) that includes a first interaction of GSH and GSNO to the G-site followed by a slower but thermodynamically favored bimolecular interaction of the free enzyme with DNDGIC. GSTM2-2 shows a small additional fluorescence perturbation that occurs after GSH binding and before DNDGIC binding. This unknown event, which probably reflects a pre-complex formation, has not been detailed because the exclusion of this additional event in the minimal reaction Scheme 2 gives a satisfactory fit to all the experimental traces. Because of its very slow rate, the interaction of GSTT2-2 with DNDGIC can be followed by a traditional spectrofluorometer (Fig. 5). Even in that case, the experimental traces fit well to Scheme 2, and this analysis confirms relatively low k on values for both the high and low affinity G-sites (3.0 ϫ 10 3 and 4.0 ϫ 10 2 M Ϫ1 s Ϫ1 , respectively). The calculated microscopic kinetic constants and the overall equilibrium constants for all representative GST members shown in Table I are in good agreement with those obtained from inhibition data at equilibrium and from static fluorescence experiments.
EPR Data-The low resolution spectrum of free DNDGIC at room temperature shows a single symmetrical line at g ϭ 2.03, typical of the rapid isotropic motion of a small molecule tumbling freely in aqueous solution (Fig. 6). Under high resolution conditions the single line can be seen to be a multiline signal; the spectrum is composed of 25 lines due to the hyperfine coupling of a single unpaired electron to 2 eq nitrosyl nitrogens and 4 eq methylene protons from the cysteines of the two bound glutathiones (Fig. 6, inset), in analogy to the similar dinitrosyldicysteinyl-iron complex (49). The EPR spectra of dinitrosyliron complexes in biological systems reported in the literature have almost always been obtained using frozen samples, typi-cally measured at 77-100 K in order to take advantage of the enhanced instrument sensitivity at low temperatures. However, under these conditions both protein-bound and free dinitrosyl complexes are immobilized and give broad anisotropic signals covering approximately the same spectral range, making it difficult to single out the individual components of a sample containing different DNIC species. We have therefore carried out experiments at room temperature where the spectra of free and bound complexes can easily be distinguished.
When substoichiometric amounts of DNDGIC are added to GSTA1-1, only the spectrum of an enzyme-bound complex is observed (Fig. 6). Binding of the complex is too fast to be measured and is completed within the experimental dead time of less than 30 s. No free complex can be detected until more than 0.5 eq of DNDGIC per subunit is added. At higher stoichiometries only part of the complex binds to the second active site, and the signal of the free complex appears in the spectrum (Fig. 6). This result confirms that binding of the complex to one active site with high affinity drastically lowers the affinity for binding at the second active site. The spectrum of DNDGIC bound to the low affinity G-site appears to be identical to that of the high affinity active site (Fig. 6, curve 4). GSTP1-1 and GSTM2-2 give very similar results, demonstrating that the presence of a high affinity binding site for a dinitrosyl-iron complex is maintained in these three classes of GST, despite differences in their substrate specificity and active site geometry. Also the GSTT2-2 isoform is able to bind the dinitrosyl- iron complex with high affinity, but in this case the rate of binding is extremely slow. Interestingly, the spectrum of the GST-bound dinitrosyl-iron complexes changes appreciably according to the GST isoform used (Fig. 7). The A1-1 and M2-2 GSTs give essentially axial spectra, whereas P1-1 and T2-2 give strongly rhombic spectra, but there are minor differences in the spectra that in practice make it possible to identify the type of GST involved directly from the EPR spectrum. The g values were determined through an anisotropic simulation of the spectra (Table II); all four complexes show almost identical g x values, whereas g y and g z values vary. The g x transitions are well separated from the others, and computer analysis reveals they all have identical line widths and line shapes; this suggests that the differences observed in the spectra are caused by geometry changes and not by ligand substitution.
Radiometric Experiments-Previous findings (18) indicated that in the active site of GSTP1-1, after binding of DNDGIC and removal of the excess of GSH by passage through a G-25 Sephadex column, the iron ion is coordinated by one GSH, two NO, and the hydroxyl group of Tyr-7. It was unclear whether a second GSH molecule could act as a fifth ligand in the presence of an excess of GSH. In fact, subtle UV-visible and EPR spectral changes have been observed previously for the bound DNDGIC when the excess of GSH is removed by gel filtration (18). In an attempt to quantify the GSH molecules present in the GSTbound complex, we adopted a well known radiometric approach used to characterize the classical ligand-receptor interaction and based on rapid filtration of a radioactively labeled ligandreceptor mixture. The excess of labeled reagent is removed by filtration, whereas the ligand-protein complex is retained by the filter. This procedure has been used for GSTA1-1 (4 M) after incubation with [ 3 H]DNDGIC (2 M) in the presence of 0.8 mM [ 3 H]GSH. The amount of the bound DNDGIC (more than 90%) has been evaluated from the extent of the final inhibition. After rapid filtration, the recovered radioactivity bound to the enzyme indicates a strict one to one stoichiometry between GSH and the bound complex. Thus, the second GSH molecule of DNDGIC is no longer present in the bound complex (at 10 mM GSH) or is characterized by a very labile interaction with the iron atom. A possible explanation of the observed EPR spectral modification observed upon removal of the excess of GSH (18) could be found in a structural modification of the DNDGICbound subunit caused by the loss of GSH bound to the uninhibited active site of the adjacent subunit.
DNDGIC May Shift from GSTM2-2 to GSTA1-1-GSTM2-2 shows a specific activity about two times higher than GSTA1-1 when CDNB is used as co-substrate (200 and 80 units/mg, respectively). Conversely, GSTA1-1 can utilize efficiently NBD-Cl as second substrate (40 units/mg), whereas this com- At high resolution the hyperfine splitting pattern of free DNDGIC could be observed in the central peak of spectrum 3 (not shown). The spectrum 3, subtracted from the spectral component of the free complex, reflects a 100% occupancy of DNDGIC to the high affinity G-site and 50% to the low affinity G-site (spectrum 4). The spectrum 4 shows a similar shape and maximum as in spectra 1 and 2 (50 and 100% occupancy of DNDGIC to the high affinity G-site) suggesting a structural equivalence of the two G-sites after binding of the complex.

TABLE II Magnetic parameters (g-tensor) for DNDGIC bound to different GST isoenzymes
The values were determined through anisotropic simulation of EPR spectra measured at room temperature (22-25°C); samples were prepared as in Fig. 7. Spectra were simulated using the same set of linewidth parameters: Lw x ϭ Lw y ϭ 8 G, Lw z ϭ 12 G. GST  pound is a poor substrate for GSTM2-2 (3 units/mg). Based on the very different specific activities toward these substrates, it was possible to verify if DNDGIC, initially bound to GSTM2-2, can be transferred to the higher affinity GSTA1-1. The experiment reported in Fig. 8 clearly demonstrates that this really occurs. When GSTA1-1 is incubated in the presence of a hemicomplexed GSTM2-2 with DNDGIC, a time-dependent decrease of activity with NBD-Cl is observed, with a concomitant increase of activity with CDNB. This is direct evidence that DNDGIC is really transferred from the low affinity GSTM2-2 to the more strongly binding Alpha class GSTA1-1. The process shows a t1 ⁄2 of about 3 min, compatible with the k off of DNDGIC from GSTM2-2 (1-2 min).
Molecular Modeling- Fig. 9 shows molecular models of the GSH-dinitrosyl-iron complex in the active sites of the four different isoenzymes. As already shown for GST P1-1 (18), the Tyr residue conserved in the A1-1, M2-2, and P1-1 isoforms (1) is in the appropriate position to act as the fourth ligand of the iron atom (together with the GSH sulfur atom and the two NO molecules), probably displacing the second GSH.
In this geometry, the complex is partially exposed to the solvent and fits perfectly to the active site, because no van der Waals violations are observed and the total interaction energy is very favorable. In particular, Ϫ200, Ϫ240, Ϫ120, and Ϫ300 kcal/mol have been calculated for GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2, respectively. These values represent the sum of Coulomb and van der Waals interaction terms between ligand and protein in the binary complex, and they do not take into account the interactions of these molecules with water (neither in the bound nor in the free state). The corresponding energies for GSH binding are similar, suggesting that the large difference in binding constants observed for the two ligands (GSH and DNDGIC) is ascribable mainly to the formation of the bond between the hydroxyl moiety of the Tyr or Ser side chain and the iron atom of the complex. This would explain the lack of affinity observed for the bacterial GST which has no Ser or Tyr residues at hydrogen bonding distance of the sulfur atom of GSH. The GSH structure is very similar in the models of the complex in the active sites of GST A1-1, M2-2, and P1-1 and does not differ appreciably from the conformation observed in the crystallographic enzyme-GSH structures. On the other hand, the GSH molecule adopts a significantly different conformation in the T2-2 isoenzyme, and the complex is mostly obscured by the extra C-terminal extension of about 40 residues which is a peculiar structural feature of this enzyme (16). However, both GSH and DNIC appear greatly stabilized in the G-site of GSTT2-2 giving even lower energy than that found in the more recently evolved GSTs. The partial exposition of the iron atom in the Alpha, Pi, and Mu class GSTs allows the presence of a second GSH molecule as a fifth iron ligand without relevant van der Waals violations (not shown), but this additional ligand cannot be located in the G-site of GSTT2-2 due to steric hindrance. DISCUSSION DNDGIC behaves like a tight binding inhibitor for all members of the human GST superfamily tested, i.e. GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2. A first kinetics approach, which did not take into account the existence of slow-reacting species, gave an overestimation of the affinity of these enzymes for DNDGIC with apparent K i values in the range of 10 Ϫ10 -10 Ϫ12 M. An overestimated K i Յ10 Ϫ11 M has been also reported previously for GSTP1-1 (18). More appropriate inhibition experiments under equilibrium conditions give reliable K i values that range from 10 Ϫ10 M for GSTA1-1, 10 Ϫ9 M for GSTM2-2, and GSTP1-1 to 10 Ϫ7 M for GSTT2-2 (Table I). Close to these values are the thermodynamic dissociation constants calculated from static fluorescence experiments (Table I) which also indicate that the binding of the co-substrate CDNB does not affect this process. These K i values are far from those reported for other GST inhibitors; a lot of GSH derivatives act as competitive inhibitors for GSTs, but their dissociation constants for the G-site are only in the range of 10 Ϫ4 to 10 Ϫ5 M (50). Thus, to our knowledge, DNDGIC represents the most potent competitive inhibitor of GSTs acting on the strictly conserved G-site.
All inhibition and fluorescence data at equilibrium indicate also the presence of a high and a low affinity binding site in these GSTs. Because of the homodimeric structure of all these enzymes, negative cooperativity triggered by DNDGIC binding is likely involved. This is the first evidence of a common intersubunit communication operating in the GST superfamily which, in this case, lowers 10 -100 times the affinity for the complex in the vacant subunit. Interestingly, in half-site complexed GSTs both the affinity of the adjacent G-site for GSH and its catalytic competence are unchanged, except for GSTA1-1 which lowers its catalytic activity of the second subunit to about 10%. Possibly unproductive binding of GSH causes such overinhibition.
Stopped-flow data fit well to a minimal binding mechanism common to all these GSTs (see Scheme 2), which includes a first interaction with GSH, according to the binding mechanisms described previously (42,43), and a slower bimolecular interaction of DNDGIC with the high and low affinity G-sites (Table I). The overall K D values calculated from the kinetic experiments are close to those given by equilibrium experiments (Table I). A careful examination of the microscopic rate constants gives additional information. It appears that DNDGIC binds to GSTA1-1, GSTM2-2, and GSTP1-1 with k on values similar to those found for GSH (10 6 -10 7 M Ϫ1 s Ϫ1 ) (42), suggesting that the G-site is open enough to accommodate DNDGIC without gross structural changes. This agrees completely with molecular modeling data that show DNDGIC partially exposed to the solvent and well stabilized in the G-sites of these GSTs. The observed strong affinity is likely due to the coordination of the iron atom to the phenolate group of the conserved Tyr residue of the active site (Tyr-7 in GSTP1-1, Tyr-6 in GSTM2-2, and Tyr-9 in GSTA1-1) which also causes a very slow extrusion of the complex from the G-site (low k off value). A lower k on value for DNDGIC binding appears to be the kinetic determinant of the decreased affinity of the second G-site in the half-saturated GSTs. Thus, negative cooperativity is not caused by a non-optimized geometry of iron ligands in the second G-site (which would cause an increased k off value) but FIG. 8. Shift of DNDGIC from GSTM2-2 to GSTA1-1. GSTM2-2 (2.5 M) was reacted for 5 min with 1.25 M DNDGIC in 0.1 M potassium phosphate buffer, pH 7.4. Residual activity with CDNB was 51% of the initial value. The enzyme was then added to an identical volume of the same buffer containing 2.5 M GSTA1-1. The amount of DNIC bound to each enzyme has been calculated at fixed times as described under "Experimental Procedures" and reported on the ordinate. f, DNDGIC bound to GSTA1-1; q, DNDGIC bound to GSTM2-2. The t1 ⁄2 for displacement of DNDGIC from GSTM2-2 is very similar to that of DNDGIC binding to GSTA1-1 (2.8 and 3.2 min, respectively).
probably by an increased rigidity or shielding of the second G-site triggered by DNDGIC binding to the first subunit.
Kinetics data also indicate that once the G-site has bound DNDGIC, GSH can bind only when DNDGIC leaves the G-site (k on for GSH binding ϭ k off for DNDGIC). This ruled out the possibility that the partial displacement of DNDGIC by an excess of GSH may occur through a transfer of the dinitrosyliron moiety from the bound DNDGIC to the free GSH. On the other hand, the mutual competition between GSH and DNDGIC for the G-site makes unlikely the possibility that the complex may be assembled in the active site by transfer of the dinitrosyl-iron moiety from the free DNDGIC to the bound GSH. However, an interesting observation is that DNDGIC may be easily translated from a low affinity GST enzyme (GSTM2-2) to a high affinity isoenzyme (GSTA1-1) (see Fig. 8). The kinetics of this event corresponds to the kinetics of DNDGIC release from the active site, so the exchange probably occurs by a simple binding of the released complex by the higher affinity enzyme. Finally, stopped-flow data give a rationale for the relatively scarce affinity of the Theta class GSTT2-2 for DNDGIC (K D ϭ 10 Ϫ7 M). In this enzyme, both k on and k off values of DNDGIC are small when compared with those of other GSTs, but k on is 3-4 orders of magnitude lower, whereas k off is only 10 -100 times lower. Thus, a low k on value appears to be the main determinant for the decreased affinity of GSTT2-2 for DNDGIC. This slow binding event could be due either to a shielded active site or to the necessity of a small structural change in order to allow the coordination of the iron atom by the hydroxyl group of Ser-11. Molecular modeling shows DNDGIC almost completely covered by the C-terminal extension which mostly obscures the G-site (see Fig. 9), whereas Ser-11 is in a proper position for iron coordination. The high negative value of energy for the complex in the G-site of GSTT2-2 and a low k off value (even lower than those found for the other GSTs) are convincing indications that the coordination in the G-site is still efficient. How is it possible to explain the coexistence of a relatively scarce affinity of GSTT2-2 for the complex with a good stabilization of DNDGIC in the G-site? The molecular modeling results show that the restricted dimensions of the GSTT2-2 G-site makes it difficult for DNDGIC to enter. In addition, it has been shown that in GSTT2-2 a rapid equilibrium exists between at least two G-site conformations, E 1 and E 2 (43). Only the less populated conformation (E 2 ) is efficient in GSH binding, and this situation possibly occurs also for DNDGIC binding. Thus, DNDGIC binding to GSTT2-2 may be summarized as shown in Scheme 3, 9. Molecular modeling. Molecular modeling of DNDGIC in the active site of GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2 was performed as described under "Experimental Procedures." The G-site is shown as a ribbon model, whereas crucial residues involved in the iron coordination (Tyr-9 in A1-1, Tyr-6 in M2-2, Tyr-7 in P1-1, and Ser-11 in T2-2) are shown as sticks, and the complex is reported as a "ball and stick" representation. The extra C-terminal segment which almost obscures the complex in GSTT2-2 is shown as a transparent helix. The iron atom is depicted as an orange sphere; the sulfur atom is colored yellow; and the oxygen is red. The figure was drawn using MolMol (46). It is evident that, in case of a fast but unfavorable equilibrium toward E 2 , the apparent k on value is low and the overall thermodynamic dissociation constant would be relatively high, just as we found experimentally.
The total absence of any interaction of the bacterial GSTB1-1 with DNDGIC can be easily explained. This enzyme shows a Cys residue (Cys-10) instead of Tyr or Ser residues in a proper distance from the sulfur atom of GSH (16,51). In addition, the native enzyme displays an unusual mixed disulfide involving Cys-10 and the bound GSH which rapidly exchange with a second GSH molecule transiently present in the G-site (52,53). Thus no thiolate groups can act as ligands for the iron atom of DNDGIC.
Radiometric and EPR data provide more information about the way DNDGIC is bound to GSTs. In fact, experiments of rapid filtration after [ 3 H]DNDGIC incubation show that a second GSH molecule is not present as the fifth ligand of the iron in the G-site. So far no crystal structures of paramagnetic DNICs have been reported, but the free complex with two NO and two GSH ligands is believed to be pseudo-tetrahedral and gives a highly axial EPR spectrum in solution when immobilized at low temperatures (18). Surprisingly, the EPR spectra of the GST-bound dinitrosyl-iron complexes are different for all the GST isoforms studied. These differences indicate changes in the geometry of the ligand arrangement around the iron or in the nature of the ligands themselves but not in the number of ligands (four). Thus, assuming a four-coordinated iron with the ligand set [N, N, S Ϫ , O Ϫ ] in the bound complex (as also suggested by the radiometric experiments reported in this paper), the axial spectra are consistent with a flattened, relatively symmetrical tetrahedral geometry for GSTM2-2 (g y ϭ g z ) and a more asymmetrical tetrahedral geometry for GSTA1-1 (g y ϭ g z ). In contrast, the rhombic spectra of P1-1 and T2-2 (g x g y g z ) indicates a very distorted structure of the bound complex in these GST isoforms. Because the A1-1, M2-2, and P1-1 show similar K D values for DNDGIC, the actual conformation of the bound complex is therefore not important for the stability, as long as the iron is coordinated efficiently to both the glutathione and a tyrosine residue. Studies on dinitrosyliron complexes containing different ligands have shown that this type of complexes can adapt a wide range of structures, depending on the constraint induced by the two other ligands attached (54). We conclude that dinitrosyl-iron complexes are firmly bound to these GST isoforms through the glutathione thiolate-iron-tyrosinate ligand arrangement; constant S-Fe-O bond angles and bond lengths are essential for the high affinity, whereas the positions of the NO groups, which do not seem to contribute much to binding, will be determined by the space available in the active site of the enzyme. This is in agreement with the molecular modeling results as can be seen from Fig. 9.
The GSTT2-2 could be a case apart as a serine residue replaces the tyrosine residue in the iron coordination. However, EPR spectra show a geometry of the bound complex very similar to that found in GSTP1-1. Thus, the reason for the slow binding of DNDGIC must be found in the difficult access to the G-site and not in a scarce stabilization in the active site, as suggested above on the basis of kinetic and molecular modeling data.
Finally, EPR data add further details concerning the cooperative mechanism. In fact, the ligand configurations of DNDGIC are identical in the hemi-saturated and in the fully saturated enzyme, because occupation of the second site does not give rise to the appearance of a second species in the EPR spectrum (see Fig. 6). The sequential mechanism (KNF) is the sole model that accounts for negative cooperativity. This model assumes a symmetric structure in the fully complexed enzyme but an asymmetric state (T-R) in the hemi-complexed enzyme. However, this situation is not confirmed by our EPR data. Alternatively, negative cooperativity may be explained on the basis of a change of flexibility or accessibility of the G-site. In that case, binding of the ligand to the first subunit would lower the accessibility to the free adjacent subunit with consequent lowering of affinity but without changing the topography of the active site. This possibility agrees well with our kinetics and EPR results.
The data reported in this paper allow interesting conclusions from an evolutionary point of view. It is accepted that the Theta class GSTT2-2 is close to the ancestral precursor of all GSTs (43), whereas GSTA1-1, GSTM2-2, and GSTP1-1 are believed to be more recently evolved enzymes. It appears that the GST superfamily is under evolutionary pressure in the direction of the optimization of the binding process of DNDGIC, and this may be related to the increasing physiological relevance of nitric oxide during evolution. In fact, only eukaryote organisms are able to use NO efficiently as chemical messenger, although the role of NO in prokaryotes remains to be solved.


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Structural insights into regulation of nuclear receptors by ligands

Nuclear receptors are DNA-binding transcription factors, the transcriptional function of many of which depends on the binding of ligands, a feature that distinguishes nuclear receptors from other transcription factors. This review will summarize recent advances in our knowledge of the interaction between selected nuclear receptors and their cognate ligands.

Introduction
Nuclear receptors are DNA-binding transcription factors. The transcriptional function of most nuclear receptors depends on the binding of ligands, a feature that distinguishes nuclear receptors from other transcription factors. When bound to an activating ligand, nuclear receptors recruit coactivator proteins such as the Steroid Receptor Coactivators (SRC-1, 2, and 3) and the TRAP/DRIP mediator complex via their LXXLL sequence motifs [McKenna et al., 1999]. In contrast, the binding of an antagonist destabilizes nuclear receptor/coactivator complexes, instead promoting interactions with corepressors such as Nuclear CoRepressor (N-CoR) and Silencing Mediator for Retinoid and Thyroid hormone receptors (SMRT) [Hu and Lazar, 2000].
Coactivators generally promote gene transcription, whereas corepressors repress gene transcription. Coactivators and corepressors are thus the Yin-Yang factors that determine the transcriptional functions of nuclear receptors and subsequent ligand-mediated physiological responses. The molecular mechanisms of ligand-mediated recruitment of coactivators or corepressors are highly conserved across the nuclear receptor superfamily. In this review, we illustrate the basic principles of these mechanisms with X-ray structures of Peroxisome Proliferator-Activated Receptor-α (PPAR-α) in complex with either an agonist and coactivator or an antagonist and corepressor.

PPAR-α
PPAR-α is a key factor in fatty acid metabolism, and is responsible for mediating the lipid-lowering effects of fibrate drugs [Issemann and Green, 1990]. As a nuclear receptor, the PPAR-α LBD contains both the ligand binding and the ligand-dependent transcription activation function (AF-2), which is located at the extreme C-terminal end of the domain. The crystal structure of the PPAR-α LBD ( Figure 1A) bound to a potent agonist, GW409544, reveals that the domain is mainly composed of α -helices that are folded into a three-layer helical sandwich [Xu et al., 2001]. The middle layer of helices is present in the upper half of the domain, but absent from the lower half of the domain, an arrangement that creates the ligand-binding pocket. Despite the conservation of the overall fold in nuclear receptors, the size and shape of the ligand binding pocket varies greatly from receptor to receptor, thus accounting for the specificity of ligand recognition and the corresponding signaling pathways.

GW409544 agonist and PPAR-α
GW409544 is a synthetic acid that activates PPAR-α with an EC50 of 5 nM [Xu et al., 2001]. The PPAR-α LBD structure reveals that the acidic group of GW409544 makes a direct hydrogen bond with tyrosine-464 of the C-terminal AF-2 helix (red helix in Figure 1A), thereby locking the AF-2 helix in the active conformation. The same mode of receptor activation is also observed with PPAR γ and PPAR Δ [Gampe et al., 2000;Nolte et al., 1998;Xu et al., 1999]. In the case of PPAR γ, mutations that destabilize the active conformation of the AF-2 helix result in severe type II diabetes and dyslipidemia. Interestingly, this defect can be rescued by a potent PPAR γ agonist, which helps to re-stabilize the active AF-2 conformation [Kallenberger et al., 2003]. These results highlight the important role of ligands and the AF-2 helix in nuclear receptor regulation.
When the AF-2 helix is positioned in the active conformation, it contributes a conserved glutamate residue to form an essential part of a "charge-clamp" pocket with a lysine from helix 3. The coactivator binds to nuclear receptor through an LXXLL motif, which adopts a two-turn α -helix (gold helix in Figure 1A) with the hydrophobic leucine side-chains docking into the hydrophobic pocket between the two charge-clamp residues. The docking of the coactivator helix is further stabilized by helix-capping interactions with the charge-clamp residues at both ends of the helix. This same mode of coactivator binding was also observed in the previous structures of the PPAR γ homodimer [Nolte et al., 1998], the RXR/PPAR γ heterodimer [Gampe et al., 2000], and receptors for thyroid hormone and estrogens [Darimont et al., 1998;Shiau et al., 1998], indicating that the charge clamp pocket is the common mechanism for coactivator recruitment by nuclear receptors.

GW6471 antagonist and PPAR-α
Corepressors bind to nuclear receptors through an LXXXIXXXL/I sequence motif. This is similar to the LXXLL motif of coactivators, suggesting that corepressors might utilize a binding mode similar to that of coactivators [Perissi et al., 1999]. Expecting that corepressor recruitment to PPAR-α would be enhanced by an antagonist, we used knowledge from agonist-bound PPAR structures to design an antagonist, GW6471, to facilitate crystallization with SMRT [Xu et al., 2002]. The chemical structure of GW6471 is similar to that of GW409544 except that the carboxylate group of GW409544 has been modified into an amide group in GW6741 ( Figure 1C). In its complex with PPAR-α, the GW409544 carboxylate makes a key interaction with Tyr464 in the AF-2 helix that is critical for receptor activation. The ethyl amide group in GW6471 was designed to block this key hydrogen bond, and to protrude into the volume normally occupied by Tyr464, thereby displacing the AF2 helix (green helix in Figure 1B and D). This prevents activation, and converts the compound from an agonist into an antagonist, greatly promoting the binding of corepressors to PPAR-α.
The crystal structure of the PPAR-α LBD bound to the antagonist GW6471 and a SMRT corepressor motif reveals several general features of antagonist-mediated assembly of the receptor/corepressor complex. First, the corepressor motif adopts a three-turn α -helix (yellow helix in Figure 1B), instead of the two-turn helix observed for coactivator motifs. Second, the corepressor-binding site overlaps the coactivator-binding site, with the additional helical turn of the corepressor motif extending into volume normally occupied by the AF-2 helix if it were in the active conformation. This provides a simple mechanism that makes coactivator and corepressor binding mutually exclusive and dependent on the position of the AF2 helix. Third, the binding of GW6471 pushes the AF-2 helix out of its active position, opening a larger binding grove to accommodate the extended corepressor helix. The repositioning of the AF-2 helix reinforces the importance of the highly flexible nature of this helix, allowing its conformation to be modulated by the binding of small molecules, either agonist or antagonist, to activate or to repress transcription.
The X-ray structure shows that SMRT makes key interactions with PPAR-α through the side-chains of its L+1, I+5 and L+9 residues. Alanine scanning mutagenesis through the SMRT corepressor motif confirms that these three core residues are the most critical for corepressor binding to PPAR-α and TR, especially the I+5 residue.
In addition, the PPAR-α residues that make up the corepressor binding pocket are also highly conserved across the nuclear receptor family (Figure 2), and the PPAR-α structure may provide a model for understanding ligand-mediated recruitment of coactivators and corepressors in other nuclear receptors.

Conclusion
In summary, ligand-dependent regulation of nuclear receptors is mediated through the highly mobile AF-2 helix, the position of which determines the ability of nuclear receptor to recruit coactivators or corepressors. Both coactivators and corepressor use a short helical motif to interact with nuclear receptors. In the presence of an agonist, the AF-2 helix is stabilized in the active conformation that forms a charge-clamp pocket to facilitate the binding of coactivator helix. In contrast, the binding of an antagonist keeps the AF-2 helix out of the active position, resulting in a larger pocket that destabilizes coactivator binding but fits better with the three-turn α -helix formed by the corepressor motif. The AF-2 helix thus serves as a ligand sensor to regulate nuclear receptor function. Furthermore, the ability to modulate the AF-2 helix by ligands has provided a great opportunity to design various ligands to control the biological processes mediated by the receptors and their related disease conditions.


extfieldsofstudy: Biology Medicine

text:
Full activation of estrogen receptor alpha activation function-1 induces proliferation of breast cancer cells.

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) alpha and beta, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptor-mediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ERalpha. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ERalpha to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ERalpha was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ERalpha and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ERalpha. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen.

The effects of estrogen and anti-estrogen are mediated through the estrogen receptors (ER) ␣ and ␤, which function as ligand-induced transcriptional factors. Recently, one of the phthalate esters, n-butylbenzyl phthalate (BBP), has been shown to induce estrogen receptormediated responses. By using the truncated types of ER mutants, we revealed that activation function-1 (AF-1) activity was necessary for the BBP-dependent transactivation function of ER␣. AF-1 is also known to be responsible for the partial agonistic activity of tamoxifen. Whereas tamoxifen exhibits an anti-estrogenic effect on proliferation of the MCF-7 breast cancer cell line, BBP showed an estrogenic effect on MCF-7 to stimulate proliferation. In vivo and in vitro binding assays revealed that whereas 4-hydroxytamoxifen (OHT) induced binding of ER␣ to both an AF-1 coactivator complex (p68/p72 and p300) and corepressor complexes (N-CoR/SMRT), BBP selectively enhanced the binding to the AF-1 coactivators. We also showed that the transcriptional activity of OHT-bound ER␣ was modulated by the ratio between the AF-1 coactivator and corepressor complexes. Expression of a dominant-negative type of N-CoR inhibited the interaction between OHT-bound ER␣ and N-CoR/SMRT and enhanced the transcriptional activity of OHT-bound ER␣. Furthermore, the cell growth of MCF-7 stably expressing the dominant-negative type of N-CoR was enhanced by the addition of OHT. These results indicated that fully activated AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen.
Estrogen is known to stimulate hormone-dependent tumors such as endometrial cancer and breast cancer (37). Recently, it was suggested that some endocrine disrupters may also contribute to the development of hormone-dependent cancers (38,39). Several studies have demonstrated that many endocrine disrupters, such as butylbenzyl phthalate (BBP), a phthalate ester used as a plasticizer, are capable of interacting with estrogen receptors and induce estrogen receptor-mediated responses, suggesting that estrogenic or anti-estrogenic effects elicited by these substances may be receptor-mediated (38 -41).
For the endocrine therapy of these cancers, the development of inhibitory ligands for ERs has yielded important therapeutic treatments, including the use of tamoxifen (37,42,43). Tamoxifen exhibits a wide range of estrogen-like and anti-estrogen actions according to the target tissue examined (44). While tamoxifen may exert anti-estrogenic activity by silencing the transcriptional activity of AF-2, agonist activity of tamoxifen can be mediated through AF-1 in a cell-or tissue-dependent manner (45)(46)(47)(48)(49). However, most patients undergoing long-term treatment of breast cancer with tamoxifen eventually experience recurrence of tumor growth. One of the reasons for this treatment failure is the acquisition by the tumor of the ability to respond to tamoxifen as a stimulatory rather than inhibitory ligand (50 -53). Wolf et al. (54,55) identified a mutant ER␣ from a tamoxifen-stimulated tumor that contained a point mutation that led to a tyrosine for aspartate substitution at amino acid 351 (ER␣(D351Y)), located within the LBD of ER␣ (54,55).
Recent studies (56 -60) have suggested that tamoxifen promotes the binding of ER␣ to nuclear receptor corepressor (N-CoR) or related factors silencing mediator of retinoid and thyroid receptors (SMRT), which mediate repression by recruiting histone deacetylases (HDACs). We reported previously (61) that ER␣(D351Y) exhibited reduced interaction with N-CoR and SMRT in the presence of 4-hydroxytamoxifen (OHT). These observations raised the possibility that OHT-dependent interaction between ER␣ and corepressor complexes may be essential for the anti-estrogenic effect of OHT and that abrogation of OHT-dependent binding of corepressors to ER␣ may convert OHT from antagonist to agonist to stimulate cancer growth.
In this paper, we identified BBP as an agonist for AF-1 of ER␣. Although BBP exhibited the same properties as tamoxifen in a transient transfection assay, BBP-occupied ER␣ did not bind to corepressors and enhanced the proliferation of the breast cancer cell line MCF-7. The transcriptional activity of OHT-occupied ER␣ was modulated by the ratio of the expression levels between AF-1 coactivators and corepressors. Moreover, MCF-7 breast cancer cell lines expressing the dominantnegative type of N-CoR exhibited a growth phenotype in the presence of OHT. These results indicate that activation of AF-1 induces the stimulation of breast cancer growth and that the ratio between AF-1 coactivators and corepressors plays a key role to prevent proliferation of tumor by tamoxifen.
Measuring IC 50 Values of E2 and BBP-For measurement of the binding constant value of BBP to ER␣, IC 50 measuring kit was purchased from Wako Chemicals Co., and IC 50 was examined according to the manufacturer's protocol.
Transfection, Luciferase Assay, Mammalian Two-hybrid Assay, and Repression Assay-293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Two days before transfection, medium was changed to phenol red-free DMEM containing 5% charcoal-stripped FBS. Transfection was performed with Lipofectin reagent (Invitrogen) according to the manufacturer's protocol. For luciferase assays, 250 ng of ERE3-tk-luc plasmid was cotransfected with 25 ng of ER expression vector (HEG0/ERG␤0) or mutants. For mammalian two-hybrid assays, 250 ng of ERE3-tk-luc or 17m5-luc vector was cotransfected with 250 ng of HEG0, GAL-DEF, or GAL-DEF(D351Y) in combination with 250 ng of indicated VP16-conjugated constructs and/or p300, p68, N-CoR, or N-CoR ID1-2 plasmids. For repression analysis, 1 g of MH100-tk-luc vector was cotransfected with 250 ng of GAL-DEF. As a reference plasmid to normalize for transfection efficiency, either 5 ng of pRL-CMV vector (Promega) or 125 ng of pRSV␤GAL vector was cotransfected in all experiments. Six hours after transfection, culture medium was replaced with fresh medium containing 0.2% FBS. At this time, either E2 (10 nM), OHT (100 nM), ICI182,780 (100 nM), ethanolic vehicle, or 5 ng/ml trichostatin A (TSA) was added, and cells were incubated for an additional 24 h. Preparation of cell extracts and luciferase assays were performed following the manufacturer's protocol (Promega). ␤-Galactosidase activity was measured to control the efficiency for each transfection. Individual transfections, each consisting of triplicate wells, were repeated at least three times. For establishing MCF-7 stable transfectant of N-CoR ID1-2, Lipofectin reagent was used for introduce pcDNA-ID1-2, and transfectants were selected by 500 g/ml G418 (Sigma), and several clones were isolated.
GST Pull-down Assay-For GST pull-down assays, bacterially expressed GST fusion proteins or GST bound to glutathione-Sepharose 4B beads (Amersham Biosciences) were incubated on ice with [ 35 S]methionine-labeled proteins expressed by in vitro translation using the TNTcoupled transcription-translation system (Promega). After 1 h of incubation, free proteins were removed by washing the beads 5 times with phosphate-buffered saline containing 10% glycerol and protease inhibitors (1 g/ml aprotinin, 1 g/ml leupeptin, and 1 M phenylmethylsulfonyl fluoride). Specifically bound proteins were eluted by boiling in SDS sample buffer and analyzed by 6% SDS-PAGE. After electrophoresis, radiolabeled proteins were visualized by autoradiography.
Cell Proliferation Analysis-Two days before assay, MDA-MB-231 (ER␣-negative) and MCF-7 (ER␣-positive) cells were cultured in a 24well plate in phenol red-free DMEM supplemented with 0.2% charcoalstripped fetal bovine serum. As experimental medium, either E2 (10 nM), OHT (1 M), BBP (1 M), or ethanol vehicle was supplemented. Cells were harvested for the indicated times, and the number of viable cells was counted with hemocytometer.
S-phase Entry Analysis-For S-phase entry analysis, NIH3T3 cells were cultured in phenol red-free DMEM supplemented with 5% charcoal-stripped FBS and seeded onto glass coverslips at 60 -70% confluence. Transfection with 3 g of wild-type or mutant ER␣ expression plasmid was performed by using Perfectin Reagent (Gene Therapy Systems) according to the manufacturer's protocol. Incubation medium was changed after 24 h into phenol red-free DMEM supplemented with 0.2% charcoal-stripped FBS. Cells were left in this medium for 24 h and then cultured with 100 M 5-bromouridine 5Ј-triphosphate (BrdUrd) in the presence of either E2 (10 nM), BBP (1 M), or OHT (1 M) for an additional 24 h. After incubation, the cells were fixed for immunostaining.

BBP Binds Ligand-binding Pocket of ER␣ and Induces the
Transcriptional Activity of AF-1-It has been reported that BBP binds to ER␣ and enhances the transcriptional activity of ER␣. To confirm the binding of BBP to ERs, we performed an in vitro competitive ligand binding assay to investigate the abilities of BBP to compete with E2 for binding to ER␣ and -␤. E2 exhibited an IC 50 of ϳ1.0 nM to both ERs (Fig. 1B), which is within the range of previously reported IC 50 values (40,65). The IC 50 values for BBP to ER␣ and ER␤ were ϳ50 M, indicating that BBP weakly bound to both receptors (Fig. 1B). Therefore, we next evaluated the ability of BBP to induce an ER-mediated response by measuring luciferase activity using 293T cells that were cotransfected with a luciferase reporter plasmid bearing estrogen response elements (EREs) and either ER␣ expression vector (HEG0) or ER␤ expression vector (ERG0␤). The results in Fig. 1, C and D, show that BBP treatment of 293T cells transiently transfected with ER␣ caused a concentration-dependent increase in luciferase activity. However, the transcriptional activity of ER␤ was not enhanced by BBP treatment (Fig. 1C). E2 treatment of either ER␣-or ER␤-transfected cells significantly induced luciferase activity (Fig. 1C). Induction of luciferase activity by BBP in transiently transfected 293T cells was completely abolished if the ER␣ construct, HEG0, was not cotransfected. In addition, 100 nM of the "pure ER antagonist" ICI182,780 completely inhibited BBP-induced transcriptional activity of ER␣ (Fig. 1D, lanes 5 and 6). These results indicate that BBP acts as an ER␣-selective agonist. To investigate the reason why BBP stimulates the transcriptional activity of ER␣ but not ER␤, BBP-dependent transcriptional activity of truncated types of ERs ( Fig. 2A) was estimated by luciferase assay. E2 enhanced the transcriptional activity of full-length ER␣ and a truncated type of ER␣, HE19 (Fig. 2B, lanes 2 and 6), which does not have an A/B region and exhibits no AF-1 activity. In contrast, BBP stimulated only the transcriptional activity of full-length ER␣ (Fig. 2B), suggesting that AF-1 is essential for the BBP-dependent transcriptional activity of ER␣. It is known that the non-steroidal anti-estrogen tamoxifen, which is the most commonly used endocrine in the treatment of all stages of breast cancer in both pre-and postmenopausal women, also exhibits AF-1 agonistic activity (45)(46)(47)(48)(49). In this experiment, OHT induced full-length ER␣-mediated transcriptional activity (Figs. 1C and 2B) but did not stimulate the transcriptional activity of HE19 (Fig. 2B), as expected. The transcriptional activity induced by BBP was about three times higher than that induced by OHT (Fig. 2B, compare lane 3 to 4). These results raise the possibility that BBP may possess the same biological properties as tamoxifen. Fluorescein-labeled estrogen bound to ERs was displaced competitively by increasing concentrations of E2 (squares) or BBP (triangles), and the ratio of displacement was measured using the luminescence meter. Displacement curves were obtained for E2 and BBP. The data were performed in triplicate and result from a representative experiment of the three repeated experiments. C, BBP enhances the transcriptional activity of ER␣. 293T cells were transiently transfected with a plasmid expressing ER␣ (HEG0) or ER␤ (ERG0␤) and a reporter plasmid bearing 3ϫ ER-responsive elements (ERE3-tk-luc) and incubated for 24 h with 10 fM to 100 M of either E2, OHT, or BBP. Transcriptional activity of ERs was measured by luciferase assay. Bars indicate fold change in luciferase activity relative to that of no ligand. Results represent the average of three independent experiments; error bars indicate S.D. D, BBP-dependent transactivation of ER␣ is inhibited by ICI treatment. 293T cells were transfected with a plasmid expressing ER␣ (HEG0) or ER␤ (ERG0␤) and ERE3-tk-luc and incubated for 24 h in the presence of either E2 (10 nM), OHT (100 nM), or BBP (1 M) with or without ICI (100 nM), and transcriptional activity was measured by luciferase assay. Bars indicate fold change in luciferase activity relative to that of no ligand. Results represent the average of three independent experiments; error bars indicate S.D.
A Contrastive Effect between BBP and OHT on the Proliferation of Breast Cancer Cells-Thus, we next examined the effects of BBP on proliferation of two human breast cancer cell lines: MCF-7 and MDA-MB-231. The MCF-7 cell line expresses ERs, and its proliferation is estrogen-dependent. MDA-MB-231 cells, whose growth is not affected by estrogens, were used to control for false-positive responses. Whereas E2 induced MCF-7 cell proliferation at concentrations at 10 nM, OHT strongly inhibited the proliferation of MCF-7 cells (Fig. 2C, right panel) (41). In contrast with OHT, BBP exerted an increase in MCF-7 proliferation at 1 to 10 M, and the maximum effect represented 70% of the presence of E2 (Fig. 2C, right  panel). None of the three compounds we tested affected proliferation of MDA-MB-231, which does not express ER␣ (Fig. 2C,  left panel).
To investigate whether the AF-1 activity is necessary for the BBP-dependent cell proliferation, newly synthesized DNA was evaluated by BrdUrd incorporation method. NIH3T3 fibroblasts were transiently transfected with expression plasmid encoding either ER␣ (HEG0) or ER␣ mutant (HE19). The transfected NIH3T3 fibroblasts were made quiescent and then stimulated for 24 h with either E2, BBP, or OHT (86). BrdUrd was added to the medium together with the ligands, and its incorporation into DNA was analyzed. BrdUrd-positive cells expressing either ER␣ or HE19 were counted. The NIH3T3 cells transfected with control vector did not respond to ligand treatments. Although E2 and BBP strongly stimulated progres-sion of ER␣-transfected NIH3T3 cells toward S-phase, BrdUrd incorporation into HE19-transfected cells was enhanced by E2 but not by BBP (Fig. 2D). These results indicate that the BBPdependent S-phase entry requires the AF-1 activity of ER␣.
BBP and OHT Induce the Binding of ER␣ to AF-1 Coactivators but Not to AF-2 Coactivators-Our results indicate that both BBP and OHT are selective agonists for ER␣ AF-1 and show an antagonistic effect on AF-2, whereas BBP exhibits an estrogenic effect and OHT shows an anti-estrogenic effect on the cell proliferation. In order to reveal a reason for this discrepancy, we tested the interaction between ER␣ and coactivators or corepressors in the presence of either BBP or tamoxifen. By using a mammalian two-hybrid assay, we first evaluated the BBP-or OHT-dependent binding of AF-1 coactivators to ER␣. HEG0 was cotransfected with either VP16 transactivation domain-fused p72 (VP-p72), p68 (VP16-p68), or p300 (VP-p300) constructs into 293T cells. All of the three ligands we tested induced the interaction of ER␣ with either p68, p72, or p300 (Fig. 3A). The BBP-dependent interaction between ER␣ and AF-1 coactivators was confirmed using a coimmunoprecipitation method. By using an anti-ER␣ antibody, ER␣ was immunoprecipitated from a nuclear extract of 293T cells that were cotransfected with ER␣ and either FLAG-tagged p72, FLAG-tagged p68, or p300. Strong anti-FLAG or anti-p300 antibody binding was observed on immunoblots of anti-ER␣ immunoprecipitates from cotransfectants treated with either E2, BBP, or OHT (Fig. 3B). We then examined the binding of the DEF region of ER␣ with AF-2 coactivators. A GST pull-down assay showed that whereas E2 induced the direct binding of ER␣(GAL-DEF) to AF-2 coactivators SRC-1, TRAP220, or TRRAP, BBP and OHT abrogated the interaction between GAL-DEF and AF-2 coactivators (Fig. 3C). In a mammalian two-hybrid experiment, E2bound GAL-DEF exhibited significant binding to either SRC-1, TRAP220, or TRRAP (Fig. 3D, left panel). Conversely, neither BBP nor OHT induced the interaction between GAL-DEF and AF-2 coactivators (Fig. 3D, middle and right panels). These results were in good agreement with the observation that BBP and OHT act as antagonists for AF-2.
OHT but Not BBP Induces the Interaction between ER␣ and Corepressor Complexes-In a previous paper, we showed that OHT induces the binding between GAL-DEF and corepressor complexes to repress the basal transcriptional activity of TK promoter located downstream from GAL4-binding elements (17 ϫ 5 m) (61). This OHT-induced binding of ER␣ to corepressor complexes is reduced by the amino acid substitution at position 351 in ER␣ (ER␣(D351Y)), which is derived from tamoxifen-induced tumor (Fig. 4A) (54,55). Consistent with previous studies, OHT-bound GAL-DEF repressed the transcriptional activity of the TK promoter (Fig. 4B, lane 2). This repressive activity was inhibited by the addition of TSA, a specific histone deacetylase inhibitor (Fig. 4B, lane 5). The D351Y mutation, which exhibited decreased corepressor asso-ciation, impaired the tamoxifen-dependent repression by GAL-DEF (Fig. 4B, lane 8). In the presence of BBP, the repressive activity of GAL-DEF was not observed (Fig. 4B, lane 3), indicating that BBP-bound ER␣ would not interact with corepressor complexes.
To test the interaction between ER␣ and corepressors, a mammalian two-hybrid assay was performed. Either GAL-DEF or GAL-DEF(D351Y) was cotransfected into 293T cells with either VP, VP-SMRT, or VP-N-CoR. Whereas the interaction between GAL-DEF and corepressors was observed in the presence of OHT (Fig. 4C, middle panel), BBP-bound GAL-DEF exhibited no binding to corepressors as expected (Fig. 4C, right  panel). Consistent with previous studies, the D351Y mutation reduced the OHT-induced interaction with corepressors (Fig.  4C, lanes 10 -12) (61). The corepressor binding was further investigated by a coimmunoprecipitation method. By using an anti-ER␣ antibody, ER␣ was immunoprecipitated from nuclear extracts of 293T cells that were cotransfected with ER␣ and V5-tagged N-CoR. In the presence of OHT, anti-V5 antibody binding was observed on immunoblots of anti-ER␣ immunoprecipitates from cotransfectants (Fig. 4D, lane 5). In contrast, N-CoR was not coprecipitated with E2-or BBP-bound ER␣ (Fig. 4D, lanes 4 and 6).
The Ratio of AF-1 Coactivator/Corepressor Complexes in Cells Is an Essential Determinant for the Transcriptional Activity of OHT-occupied ER␣-Our results indicated that al-

FIG. 3. OHT and BBP induce the binding of AF-1-specific coactivators to ER␣. A, recruitment of AF-1 coactivators to BBP-or
OHT-occupied ER␣. Mammalian two-hybrid assays using ER␣, along with either VP-p68, VP-p72, VP-p300, or VP, were performed. Indicated plasmids were transfected into 293T cells with HEG0 and ERE3-tk-luc in the absence (none) or presence of either E2 (10 nM), OHT (100 nM), or BBP (1 M). Bars show fold change in luciferase activity relative to HEG0 and VP vectors in the absence of ligands. Results represent the average of three independent experiments; error bars indicate S.D. B, confirmation of OHT-and BBP-induced binding between ER␣ and p68/p72/p300 by coimmunoprecipitation. Nuclear extracts were prepared from 293T cells coexpressing ER␣ and FLAG-tagged p68, FLAG-tagged p72, or p300. ER␣ was then immunoprecipitated by anti-ER␣ antibody, and immunoprecipitates were detected by immunoblotting with anti-FLAG or anti-p300 antibody. C, BBP or OHT does not induce the binding of ER␣ to AF-2 coactivators in vitro. GST pull-down assays were performed using GST or GST-fused ER␣ DEF region produced by Escherichia coli with either [ 35  though OHT induced the binding of ER␣ to both AF-1 coactivators and corepressors, BBP-bound ER␣ selectively associated with AF-1 coactivators. Thus we next examined the effect of AF-1 coactivators and corepressors on transcriptional activity of ER␣ induced by OHT or BBP. The expression of p300 and p68 enhanced the transcriptional activity of ER␣ in the presence of E2, OHT, or BBP (Fig. 5A). On the contrary, N-CoR reduced OHT-dependent transactivation function of ER␣ (Fig.  5B, middle panel) but not E2-and BBP-dependent activation (Fig. 5B, left and right panels). The stimulation of OHT-dependent transcriptional activity of ER␣(D351Y) by the expression of AF-1 coactivators was much higher than that of wildtype ER␣ (Fig. 5A, compare left to middle panel), suggesting that the association of endogenous corepressor complexes with OHT-bound ER␣ reduces AF-1 activity. The maximum OHTdependent transcriptional activity induced by coactivator expression was comparable with the BBP-dependent activity (Fig. 5A, compare lanes 14 -16), raising the possibility that the overexpression of coactivators may convert OHT from antagonist into agonist to stimulate cancer growth. In addition, as shown in Fig. 5D, enhancement of transcriptional activity by the coactivator expression was reduced by the expression of corepressor, N-CoR. These findings indicate that the ratio of coactivator/corepressor in cells determines the transcriptional activity of OHT-bound ER␣ and that the inhibition of corepressor binding to ER␣ may convert OHT from partial agonist to full agonist for AF-1 to stimulate cell growth of MCF-7.

Inhibition of the Interaction between OHT-bound ER␣ and Corepressors Stimulates Proliferation of Breast Cancer Cells in the Presence of OHT-
To investigate this hypothesis, we first determined the regions in N-CoR/SMRT, which are responsible for the interaction between N-CoR/SMRT and OHT-occupied ER␣. Recently, it was shown that retinoid X receptor and TR LBDs associate with N-CoR/SMRT via the N-CoR/SMRT domains ID1 and ID2 (66 -69). Therefore, we assessed whether GST-ID1-2 fusion proteins could associate with in vitro translated ER␣. A GST pull-down assay showed both GST-ID1-2 derived from N-CoR and SMRT directly bound to ER␣ in the presence of OHT (Fig. 6B). We then studied whether the expression of the ID1-2 region abrogates the interaction between OHT-occupied ER␣ and N-CoR/SMRT using a mammalian twohybrid assay. In this assay, the binding of N-CoR/SMRT to OHT-bound ER␣ was inhibited by the expression of the ID1-2 region derived from N-CoR (Fig. 6C). Coexpression of the ID1-2 region with ER␣ enhanced the OHT-dependent transactivation function of ER␣ (Fig. 6D, left panel) but not that of ER␣(D351Y) . Bars indicate fold change in luciferase activity relative to the GAL-DEF and VP vectors. Results represent the average of three independent experiments; error bars indicate S.D. D, the confirmation of OHT-and BBP-induced binding between ER␣ and N-CoR by coimmunoprecipitation. Nuclear extracts were prepared from 293T cells coexpressing ER␣ and V5-tagged N-CoR. ER␣ was immunoprecipitated by anti-ER␣ antibody, and V5-N-CoR in the anti-ER␣ immunoprecipitates was detected by immunoblotting with anti-V5 antibody. (Fig. 6D, right panel), indicating that the inhibition of corepressor binding enhances the AF-1 activity of ER␣.
Next, we examined the effect of the expression of the ID1-2 region on the E2-dependent growth phenotype of the MCF-7 breast cancer cell line. The expression vector containing FLAGtagged ID1-2 region was transfected into MCF-7 cells, and several stable cell lines constitutively expressing the ID1-2 region in MCF-7 cells (MCF-7(ID1-2)) were established. We picked up four independent clones, clones 1-4, and we examined the expression of ER␣ and ID1-2 by Western blot using specific antibodies against ER␣ and FLAG epitope. In all four clones we tested, the expression of FLAG-ID1-2 was observed (Fig. 7A). The expression level of ER␣ in these clones was unchanged when compared with the control MCF-7 cells (Fig.  7A). In the absence of E2, ID1-2 stable clones exhibited normal cell growth similar to the control MCF-7 (Fig. 7B). However, either E2 or BBP treatment enhanced the growth rate of the control and clones. OHT inhibited the growth of control MCF-7 cells but stimulated the growth of ID1-2 stable transformants (Fig. 7B), indicating that the inhibition of corepressors binding to ER␣ converts OHT from anti-estrogenic to an estrogenic effect on the proliferation of breast cancer cells. DISCUSSION BBP is a phthalic ester that is present in papers and paperboards used as packaging materials for aqueous, fatty, and dry foods (38,40,41). BBP has been shown to possess estrogenic properties in vitro and in vivo (40,41). We showed that whereas BBP bound to ER␣ and ER␤, it stimulated the transcriptional activity of ER␣ but not that of ER␤. The results obtained from the experiments using the truncated type of ER␣, HE19, revealed that the A/B region of ER␣ was responsible for the transactivation induced by BBP. This property of BBP resembles the property of tamoxifen which engenders a conformational change in the ligand binding domain distinct from that induced by E2 and inhibits the activity of the hormone-dependent AF-2 but not AF-1 (70,71). Whereas both BBP and tamoxifen acted as an AF-1-selective agonist, BBP and tamoxifen exhibited a contrastive effect on proliferation of the breast cancer cell line MCF-7. OHT showed an anti-estrogenic effect on MCF-7 cells, and BBP had an estrogenic effect to stimulate proliferation of MCF-7.
The Structure of LBD Induced by BBP Is Different from That Induced by OHT-Accumulating evidence suggests that the . Indicated plasmids were transfected into 293T cells with ERE3-tk-luc in the presence of OHT (10 nM). The amount of transfected plasmids for p68/p300 and N-CoR was 0.25 (ϩ), 0.5 (ϩϩ), and 1.0 g (ϩϩϩ), respectively. Bars indicate fold change in luciferase activity relative to ER␣ in the absence of ligands. D, the ratio between AF-1 coactivators and corepressors regulates the OHT-dependent transcriptional activity. Indicated plasmids were transfected into 293T cells with ERE3-tk-luc in the presence of OHT (100 nM). The amount of transfected plasmids for p68/p300 and N-CoR was 0.25 (ϩ) and 0.5 g (ϩϩ), respectively. Bars indicate fold change in luciferase activity relative to ER␣ in the absence of ligand. differential ability of partial antagonists to modify gene expression cannot be accounted for by alterations in the ligand-receptor complex alone but also must take into consideration coregulator (coactivator and corepressor) proteins that regulate ER interaction with the general transcriptional machinery and chromatin (15,32,59,(72)(73)(74)(75). Therefore, coactivators and corepressors of the ER␣ were tested to determine whether these coregulators interact with ER␣ in the presence of these compounds. All of the three compounds we tested induced the interaction between ER␣ and AF-1 coactivators p68/p72 and p300 as expected. E2 induced the binding of LBD to AF-2 coactivators but not to corepressors (32,59,74,75,78). Consistent with previous reports (70,71), OHT induced the interaction with corepressors instead of AF-2 coactivators. In contrast, BBP-occupied LBD bound to neither AF-2 coactivators nor corepressors.
The crystal structures of the LBDs of several nuclear receptors have been determined and described as a sandwich of 12 ␣-helices (H1-H12) with a central hydrophobic ligand-binding pocket (70,71,76,77). In the presence of ligands, the hinge region between H11 and H12 is moved closer to H3 and H5, and H12 is positioned over the ligand-binding pocket formed by H3, H4, and H5. The repositioned H12 releases the corepressors from the LBD and forms a hydrophobic groove with H3 and H5 (70,71). This hydrophobic groove is known to be important for interaction with LXXLL motifs found in p160 family members (SRC-1, transcription intermediary factor 2, and p300/CBPinteracting protein) as well as in other coactivator molecules (49,71,79,80). The structures of tamoxifen-bound ER␣ re-vealed that the position of H12 differed compared with that of H12 in E2-bound ER␣ and did not form a coactivator interaction surface but a recognition surface for corepressor complexes (70,71). We observed that BBP induced the binding of LBD to neither AF-2 coactivators nor corepressors, indicating that the position of H12 in BBP-bound ER␣ is different from that of H12 in E2-or OHT-bound ER␣. These results also suggest that the structural differences induced by compound binding would affect the binding affinity of ER␣ to the coregulators.
The Ratio between AF-1 Coactivator and Corepressor Complexes in Cells Is a Major Determinant of the Transactivation of OHT-occupied ER␣-Our results indicated that OHT induced the binding of ER␣ to both AF-1 coactivators and corepressors. The enhancement of transcriptional activity induced by coactivator expression was reduced by the corepressor expression, indicating that the ratio of coactivator/corepressor in cells determines the transcriptional activity of OHT-bound ER␣. It is well known that p300 possesses histone acetyltransferase activity that modifies local chromatin structure into a transcriptionally permissive state (16,17). N-CoR/SMRT complexes contain histone deacetylase activity (62,(81)(82)(83), suggesting that AF-1 coactivator and corepressor complexes may act and/or bind competitively to tamoxifen-bound ER␣. If the OHT-occupied ER␣ simultaneously binds coactivators and corepressors under these conditions, the repressor domain of corepressors may inhibit ER␣ transcriptional activity by blocking the activation function of coactivators. Alternatively, OHT may induce competitive binding between AF-1 coactivators and corepressors to abrogate full AF-1 activity. OHT may induce an LBD conformation that enables the receptor to retain its ability to interact with corepressor to decrease the accessibility of AF-1 coactivators to the A/B region such that AF-1 activity is not efficiently activated.
Enhancement of the AF-1 Activity Stimulates Cancer Cell Growth-In a previous paper, we showed that the ER␣(D351Y) mutant, which is derived from an MCF-7 breast tumor cell line that showed stimulated growth rather than inhibition by tamoxifen, exhibited reduced OHT-dependent interaction with corepressors to increase OHT-induced AF-1 activity compared with wild-type ER␣ (61). The AF-1 activity induced by BBP was comparable with that of OHT-bound ER␣(D351Y), since BBP induced the binding of ER␣ to AF-1 coactivators but not to corepressors. These results raise the possibility that fully acti-vated AF-1 induces the proliferation of breast cancer cells and that the binding of corepressors to ER␣ is essential for the antagonistic effect of OHT to inhibit proliferation of breast cancer cells. Therefore, we generated cell lines stably expressing ID1-2 region of N-CoR to abrogate OHT-dependent binding of ER␣ to corepressors. The stable transformants expressing the ID1-2 region exhibited the OHT-stimulated growth phenotype, indicating that the binding between OHT-occupied ER␣ and corepressors plays a key role in inhibition of tumor growth by tamoxifen. In addition, these results also indicate that fully activated AF-1 stimulates cancer growth.
Tamoxifen is an effective treatment for all stages of hormone-responsive breast cancer and can prevent breast cancer in high-risk women (44,84). However, tamoxifen has a partial estrogenic activity in the uterus and is associated with an increased incidence of endometrial hyperplasia and cancer. Recently, Brown and co-workers (75) showed that the expression level of SRC-1, a coactivator for ER␣, is higher in an endometrial cancer cell line, Ishikawa, than in MCF-7. SRC-1 silencing by small interfering RNA in Ishikawa cells resulted in inhibition of tamoxifen-stimulated cell cycle progression. Hayashi and co-workers (85) reported that the relative transcriptional activity of AF-1 of ER␣ compared with that of AF-2 was 4-fold higher in Ishikawa cells than MCF-7 cells. They also mentioned that mitogen-activated protein kinase, which phosphorylates the serine residue at position 118 in ER␣ A/B domain, was constitutively activated in Ishikawa cells but not in MCF-7 cells. We reported previously (34 -36) that the phosphorylation of serine 118 by mitogen-activated protein kinase stabilized the complex formation between ER␣ and p68/p72 DEAD box proteins, which are components of the p300/SRC-1 coactivator complex, to stimulate AF-1 activity of ER␣. Together with these observations, our results suggest that the ratio between ER␣ AF-1-coactivator complex and ER␣-corepressor complex in cells is an important determinant of AF-1 activity of OHToccupied ER␣ and that the proliferation of cancer cells is regulated by AF-1 activity of OHT-bound ER␣. From these observations, it is suggested that the tissue and cell type specificity of tamoxifen action is due to the difference of AF-1 activity of tamoxifen-bound ER␣ in various tissues and cells. Our results shed light on the molecular mechanism underlying tamoxifendependent inhibition of cancer growth and ring an alarm against endocrine disrupters.