The ligand insertion hypothesis in the genomic action of steroid hormones1

PII:

J. Steroid Biochem. Molec. Biol. Vol. 65, No. 1±6, pp. 75±89, 1998 # 1998 Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain S0960-0760(97)00186-6 0960-0760/98 $19.00 + 0.00

The Ligand Insertion Hypothesis in the Genomic Action of Steroid Hormones Lawrence B. Hendry,1* Edwin D. Bransome Jr.2 and Virendra B. Mahesh1 1

Drug Design and Development Laboratory, Department of Physiology and Endocrinology CLW3134, Augusta, GA 30912, U.S.A. and 2Department of Medicine, Medical College of Georgia, Augusta, GA 30912, U.S.A.

Gene regulation by steroids is tightly coupled to hormone concentration and stereochemistry. A key step is binding of hormones to receptors which interact with consensus DNA sequences known as hormone response elements (HREs). The speci®city and strength of hormone binding do not correlate well with hormonal activity suggesting an additional step involving recognition of ligand by the gene. Stereospeci®c ®t of hormones between base pairs and correlation of ®t with hormonal activity led to the proposal that such recognition involves insertion of hormone into DNA. Here, the feasibility of insertion was investigated using computer models of the glucocorticoid receptor DNA binding domain bound to its HRE. The site reported to accommodate glucocorticoids was found in the HRE and was exposed to permit unwinding at this locus. The resulting cavity in the unwound DNA/ receptor interface ®t cortisol remarkably well; cortisol formed hydrogen bonds to both the receptor and DNA. Current experimental evidence is generally consistent with ligand binding domains of receptors undergoing a conformational change which facilitates transfer of the ligand into the unwound DNA/receptor interface. We propose this step is rate limiting and alterations in receptor, DNA or hormone which attenuate insertion impair hormonal regulation of gene function. # 1998 Published by Elsevier Science Ltd. All rights reserved. J. Steroid Biochem. Molec. Biol., Vol. 65, No. 1±6, pp. 75±89, 1998

INTRODUCTION

detect a pinch of sugar dissolved in a swimming pool'' [1]. Historically, the progression of thought leading to the now well accepted classical mechanism was in¯uenced by early experiments of Munck and colleagues in the late 1950s which demonstrated that steroid hormones could interact with nucleic acid components [2, 3]. In the case of estrogen action, Jensen recounts the prevailing thinking at that time as focused on two hypotheses [4]. One view was that ``...estrogen being a ¯at molecule, could intercalate between base pairs in nucleic acid structure and in some way alter template function or other activity of DNA''. The widely accepted hypothesis, however, was that estradiol underwent an enzymatic `transhydrogenation' and that such enzymatic conversions were the general mechanism by which steroid hormones acted. Evidence to the contrary that enzymatic conversion of steroids was not required for hormone action was provided by Bush and Mahesh using corticosteroids [5] and Jensen and Jacobson with

The genomic actions of steroid hormones to which many essential physiological functions as well as pathological conditions are critically linked have yet to be fully elucidated. These actions are particularly interesting given that unlike many natural products, minute physiological levels of hormone (10ÿ8 to 10ÿ10 M) have profound biological effects in target tissues which are speci®c for a given structure and proportional to hormone concentration. In this regard, O'Malley and Schrader made the following analogy ``If the palate were as sensitive to ¯avors as the target cells are to hormones, we would be able to Proceedings of the 13th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology ``Recent Advances in Steroid Biochemistry & Molecular Biology'' Monaco 25±28 May 1997. *Correspondence to L. B. Hendry. Present address: Pharmacogenetics Development Co., Inc. P.O. Box 1667, 1164 Broad Street, Augusta, GA 30903. Tel: 706 821 3603; Fax: 706 821 3606. 75

76

L. B. Hendry et al.

estradiol [6]. In 1960, Clever and Karlson discovered that the insect hormone ecdysone caused chromosomal puf®ng in drosophila [7] demonstrating that hormonal steroids could have pronounced effects on gene function. Remarkably close, albeit enigmatic, relationships between the structures of steroid hormones and nucleic acid base pairs were reported in 1962 by Huggins and Yang [8]. In the mid 1960s, pioneering experimental studies notably by the laboratories of Jensen [9±11] and Gorski [12, 13] led to the breakthrough discovery of intracellular proteins which speci®cally bound steroid hormones and concentrated them in physiologically responsive tissues. The steroid/protein receptor complex was clearly shown to be present in target cell nuclei and to interact with and speci®cally regulate hormonally responsive genes. Modern techniques in molecular biology have facilitated dramatic advances in our understanding of the genomic mechanism which is now known to be a complex series of highly integrated steps such as the dissociation of heat shock proteins [14], receptor phosphorylation [15] and the assembly of numerous transcription factors [16, 17]. In fact, the discovery of the involvement of multiple nuclear factors has led to the notion of tripartite ligand±receptor±effector site systems [18]. Despite this complexity, there are certain features which are common to the genomic action of steroid hormones. The hormones bind to receptors which are part of a superfamily [19] with similar structural features: a centrally located, conserved DNA binding domain (DBD), a less conserved carboxy terminal ligand binding domain (LBD) and a variable amino terminal domain. In addition to receptors for the mammalian steroid hormones, the superfamily contains receptors for ecdysone, thyroid hormone, cis- and trans-retinoic acid, 1,25-dihydroxyvitamin D3 and orphan receptors for which there are no known ligands. The receptors generally bind as homo or heterodimers to common consensus gene sequences called hormone responsive elements (HREs). The HREs are composed of closely related half sites of ®ve to six bases which bind to a single receptor monomer separated by a variable number of indiscriminate bases. The HREs are arranged in palindromes, pseudopalindromes or direct repeats which mediate their interaction with receptor dimers. Unanswered questions in the classical genomic mechanism of hormone action Although the essential role of receptors in hormone action is undisputed, the exact mechanism by which the concentration and stereospeci®city of the hormonal ligand is ultimately recognized by the gene is unknown. Under physiological conditions in vivo, hormone action does not take place in the absence of ligand even though the hormone receptor is present in abundant quantities. Under certain in vitro con-

ditions, receptors can function in the absence of ligand [16, 17], however, in cell free ligand-dependent transcription systems the addition of ligand causes impressive increases in transcriptional activity over basal levels achieved with receptor alone [20, 21]. A popular view has been that hormonal activity ultimately results from a highly speci®c conformational change in the receptor protein elicited by ligand binding. Thus, within any group of candidate ligands, the degree of receptor binding which is responsible for the conformational change in the protein should correlate with degree of hormonal activity. There is considerable evidence, however, that the degree of binding of hormone analogs does not correlate well with degree of hormonal activity [22±40]. For example in the case of estrogens, weakly binding analogs have been reported to have super potent hormonal activity, i.e. much greater than the natural hormone [23±28], while other very strong binding ligands have been found to be weakly active [30±33]. Such differences cannot be explained by the bioavailability or metabolism of various ligands. In addition, the magnitude and rank order of binding activities of estrogen analogs to the estrogen receptor are known to vary widely in different species and in different tissues [41]. Jensen and collaborators, moreover, have provided evidence that there are several distinct binding sites on the same receptor which have different af®nities for hormone and certain antagonists [42, 43]. Unreliable structure±binding af®nity correlations as well as the fact that receptor binding cannot be used to predict whether a given ligand will be an agonist or antagonist have also led other investigators to suggest there may be a second binding site [22, 44±46]. This suggestion is consistent with the ®nding that the binding of receptor to the HRE appears to be unaffected by ligand, although the ligand is required for full transcriptional activity [47±50]. Leaving aside the recent discovery of a second estrogen receptor with differences in the LBD and possible variations in ligand binding properties [51], available experimental evidence does not support the popular hypothesis that ligand binding to receptor alone elicits quantitative effects on transcription. Evidence indicating a ligand±DNA interaction That there may be an as yet unelucidated step involving recognition of the hormone by the gene could also be argued from the size and location of the molecules involved. Namely, small structural changes in the stereochemistry of small molecules with hormonal activity cause dramatic changes in transcriptional activity and must somehow be re¯ected in alterations of two relatively large macromolecules i.e. the receptor and DNA. In terms of primary sequence, the DBD of the receptor which binds to the HRE and directly affects the transcriptional machinery is distal to the hormone binding site in the LBD. Also

Ligand insertion into DNA-receptor complexes

indicative of an additional critical recognition step is the fact that the HREs which bind their cognate receptors have in many cases identical sequences. For example, TGTTCT is conserved in HRE half sites which bind glucocorticoid, mineralocorticoid, progesterone and androgen receptors. How is it then possible for the respective hormones i.e. cortisol, aldosterone, progesterone and testosterone to speci®cally activate transcription of their cognate HREs when the nucleotide sequence is identical? Perhaps the most persuasive experimental evidence that the role of hormone in the genomic mechanism goes beyond binding to the receptor is the observation that damage to DNA can be caused by hormone analogs. Nutter et al. have shown that certain estrogen analogs cause DNA single strand breaks [52]. Telang et al. found that 16a-hydroxyestrone induces genotoxic damage and have suggested this may be caused by direct interaction with cellular DNA [53]. Experiments by Liehr et al. have also shown DNA damage and putative covalent adduct formation can occur in rats treated with the synthetic estrogen diethylstilbestrol as well as with catechol estrogens [54]. DeSombre et al. reported that 123 I labeled estrogens damage DNA [55]. The damage occurs in cells containing the estrogen receptor but not in cells without receptor. These ®ndings appear to require a mechanism in which hormone can come in direct contact with the gene. The recent isolation and characterization of an adenine-estrogen adduct by Abul-Hajj et al. using electrochemical techniques clearly demonstrate that such DNA±steroid interactions are chemically feasible [56]. Albeit controversial, there is also increasing evidence that DNA adducts can be formed with certain estrogen antagonists demonstrating that these ligands can also come in direct contact with DNA [57±63]. The `ligand insertion hypothesis' and predictions which can be made from this mechanism A proposal that could address certain unanswered questions regarding hormone action was ®rst made in 1977 [64]. The hypothesis was that there was an unexplored step in gene regulation involving insertion of certain hormones into DNA facilitated by chromosomal proteins. The `ligand insertion hypothesis' as depicted in a simpli®ed cartoon (Fig. 1) does not contradict the traditional genomic mechanism of ligand±receptor±DNA interaction but suggests that the receptor can adopt a conformation which promotes the bending and unwinding of DNA thereby creating a cavity into which hormone is inserted. Thus, the strength of receptor binding of a given hormone need not correlate with hormonal activity and full recognition of a ligand structure is the result of ®t into the cavity formed in the unwound DNA/protein interface.

77

The basis of the `ligand insertion hypothesis' was the discovery initially twenty years ago of complementary stereospeci®c ®t of steroid hormones between base pairs using space ®lling models [64, 65]. Since that time, similar results have been obtained with other hormones in the superfamily [66] and the fundamental observations have been con®rmed with computer modeling and energy calculations ( [67±70] and references therein). All of the natural hormones were best accommodated by the same sequence i.e. 5'-TG-3
5'-CA-3' and each hormone possessed a distinctive pattern of stereospeci®c hydrogen bonds to DNA. It followed that if ligand insertion was taking place, several predictions could be made [66]: (1) the sequence 5'-TG-3
5'-CA-3' would be found in critical locations in genes regulated by hormones in the superfamily; (2) the sequence would be capable of unwinding and forming a cavity to accommodate hormone; (3) a correlation would be observed between hormonal activity and degree of ®t of hormones and candidate ligands into the unwound site in DNA; (4) attempts to insert certain ligands, e.g. those capable of forming covalent linkages, could damage the DNA; (5) receptors would facilitate bending and unwinding of DNA in concert with insertion of hormone at 5'TG-3
5'-CA-3' and (6) key amino acids in the receptor proteins would control the speci®city of hormonal responses by modulating the conformation of the unwound DNA and the capacity of hormone to form speci®c hydrogen bonds upon insertion into the site. These initial predictions predated full characterization of hormone regulatory sequences and the discovery of 5'-TG-3
5'-CA-3' in consensus HREs. This site is frequently located at the ends of half sites throughout the superfamily and as predicted it is closely associated with transcriptional activity [16, 17]. Given the relatively large number of HREs which have now been characterized throughout the superfamily, it would have been impossible to have arrived at these predictions by happenstance. We have also shown that hormonal activity correlates well with ®t of compounds into the unwound sequence 5'-TG-3
5'-CA-3' [67]. This observation has led to the development of pharmacophores (three dimensional blueprints) which can be used to design drugs with endocrine activity as well as to screen and identify environmental chemicals that are potential endocrine disrupters [23]. It has also now been well established that bending of DNA occurs upon binding of several receptors in the superfamily to their cognate HREs [71±81]. In cases where the position of bending has been studied, i.e. the progesterone, thyroid and retinoic acid receptors, the bend is near the center of the HRE consistent with the location of the site 5'-TG-3
5'-CA-3' where hormone can be accommodated [71, 80]. Recently, we reported that the predicted locus of insertion of the natural hormone cortisol, i.e. 5'-TG-

78

L. B. Hendry et al.

Fig. 1. Cartoon depicting a `ligand insertion' step in the genomic mechanism of hormone action in the steroid/ thyroid/vitamin D/retinoic acid superfamily: (A) the essential components; (B) the traditional mechanism in which binding of hormone to the receptor is distal to the interaction of the receptor with its cognate hormone response element (HRE); (C) a ligand insertion step in which binding of receptor to the HRE results in bending and unwinding of DNA followed by insertion of the hormone into the cavity formed in the receptor/ unwound HRE interface.

3
5'-CA-3', is uniquely exposed in the complex of the glucocorticoid receptor (GR) DBD with the glucocorticoid response element (GRE) and can be contacted by the steroid [70]. We now assess the feasibility for hormone to be inserted into DNA in the presence of the receptor. The glucocorticoid system was again chosen because it has been one of the most extensively and rigorously studied both in vivo and in vitro [50] and, detailed, consistent structural data derived from both X-ray [82] and re®ned solution NMR [83] have been reported. Unwinding the GRE in the presence of the GR DBD results in a cavity at 5'-TG-3
5'-CA-3' which ®ts the hormone and facilitates stereospeci®c hydrogen bonding unique to cortisol. In the resulting ternary complex, both the receptor and unwound DNA contribute to recognition of the cortisol molecule. In the discussion of

our ®ndings, we cite further supporting evidence of other investigators for the ligand insertion hypothesis. MATERIALS AND METHODS

Molecular modeling was conducted on a Silicon Graphics R4400 Indigo2 Extreme equipped with stereographics using Sybyl 6.04 software (Tripos Associates, St. Louis, MO). Cortisol was constructed from fragment libraries and minimized using the Sybyl force ®eld. Ent-cortisol was made by inverting the stereochemistry of cortisol. The Gasteiger±Huckel method was used to calculate sigma and pi charges on the steroids. The coordinates of the GR DBD/GRE [82], termed the wound complex, were obtained from the X-ray crystal entry 1GLU (deposited 8-30-92; revised date 1/31/94) in the Brookhaven database [84, 85]. Atom

Ligand insertion into DNA-receptor complexes

Fig. 2. Computer generated space ®lling models in stereo of: (top) the wound complex of the GRE (red) with the GR DBD (green); (bottom) the corresponding unwound complex of the GRE with the GR DBD. The ®rst two bases at the end of the speci®c half site in the GRE 5'-TG-3'
5'-CA-3' are colored cyan; Arg 466 and Lys 490 of the GR DBD are blue; hydrogen bonding functional groups which form stereospeci®c linkages are yellow (cf. Figures 3 and 4).

79

80

L. B. Hendry et al.

Fig. 3. Diagram of the unwound site 5'-TG-3'
5'-CA-3' which ®ts hormones in the steroid/thyroid superfamily in the same orientation as in Figs 2, 4 and 5. Selected heteroatoms on the DNA capable of forming stereospeci®c hydrogen bonds to hormones are numbered: 1, 6 (POÿ); 2, 7 (POH); 3, 8 (O4' of deoxyribose); 4 (HOH. . .O4 of T); 5 (N7 of G); 9 (HN6 of A). Amino acids of the GR which form stereospeci®c hydrogen bonds in the unwound GRE/cortisol/GR DBD complex i.e. Lys 490 and Arg 466 are numbered 10 (HN) and 11 (HN), respectively. The hatched line separates linkages on complementary DNA strands. The ligands are separated into groups which have the same half site sequences in their respective HREs (cf. Ref. [17]). The linkages to each hormone in the superfamily are listed. $ and * designate salt bridges and ligands not yet examined by computer modeling, respectively. Although not shown, ecdysone can also ®t into this site.

types were appropriately assigned consistent with the Sybyl program and hydrogens were added using the Biopolymer module. Charges were assigned using the Kollman All Atom option for both proteins and nucleic acids. The unwound GRE was constructed by replacing the base pairs at the end of the half site (5'TG-3'
5'-CA-3') with an unwound model of this sequence [67]. The position of the unwound GRE was adjusted to maximize interaction with the GR DBD. Docking of cortisol and ent-cortisol in the wound and unwound GR DBD/GRE complex was performed in stereo initially with the autodocking program followed by visual evaluation of the contact of van der Waals surfaces with space ®lling images and automonitoring of the distances between atoms with skeletal images. The latter was used to assess the formation of favorable electrostatic interactions e.g. potential hydrogen bonds. Connolly solvent accessible Ê surfaces derived from a probe atom radius of 1.0 A

were also employed to assess the complementary ®t of the molecular surfaces. The Sybyl force ®eld with Ê value for the van der Waals radius of hydroa 1.2 A gen was used to measure the magnitude of the interaction between the steroid and the protein/DNA complexes. Complementary ®t was quantitated in kcals by increases in steric attraction (a negative van der Waals energy) and hydrogen bonding (a negative electrostatic energy). With regard to the latter, the electrostatic interaction between donor protons and acceptor heteroatoms was measured. Adjustments were made to the conformations of rotatable bonds of the steroid, nucleic acid and protein to maximize favorable electrostatic contacts. The total changes in electrostatic and van der Waals energies were used to compare degree of complementarity. A convenient method was to de®ne the GR DBD/GRE and the steroid separately as aggregates prior to docking. Multiple docking experiments were conducted until the decrease in energy was optimized. Water molecules were considered in cases where they could readily facilitate a stereospeci®c hydrogen bond by incorporation into a complex (e.g. the 11b hydroxyl of cortisol in the GR DBD/unwound GRE complex). It should be noted that while the autodocking procedure has been a valuable tool for general assessment of the interactions of molecules [70], the absolute values of energy changes are approximations which will vary depending upon those lattice points automatically assigned to represent a given structure. The ®nal energy values reported here were derived from the exact positions of the atoms in each molecule and thus can be employed reliably to compare the relative ®t of ligands into the GR DBD/GRE.

RESULTS

Wound and unwound GRE/GR DBD complexes The wound GRE/GR DBD model (Fig. 2) contains two half sites in the GRE separated by a four base pair spacer with the GR DBD binding as a dimer; each subunit contacts separate half sites [82]. The upper subunit of the GR forms a sequence speci®c interaction with one GR DBD half site [82, 83] and was thus chosen for focus in these experiments. The ®rst two base pairs i.e. 5'-TG-3'
5'CA-3' are largely exposed in the `speci®c' half site with only the N7 and O6 of G contacting the protein via hydrogen bonds to Arg 466. Construction of the complex of the GR DBD with the unwound GRE (Figs 2 and 3) resulted in bending of the DNA. A cavity bordered by receptor was formed between base pairs at 5'-TG-3'
5'-CA-3'. The hydrogen bond between Arg 466 and the O6 of G was maintained and a new salt bridge was formed between an oxygen of the 5'-phosphate group of G and Lys 490.

Ligand insertion into DNA-receptor complexes

Fig. 4. Computer models in stereo colored as in Fig. 2: (top) skeletal model of cortisol (magenta) inserted into the unwound GRE/GR DBD interface with dotted yellow lines designating stereospeci®c hydrogen bonds; (middle) space ®lling model of cortisol inserted into the interface and (bottom) space ®lling model showing contacts of cortisol with GR DBD upon removal of the GRE.

81

82

L. B. Hendry et al.

Fig. 5. Computer generated Connolly surfaces in stereo of: (top) the unwound GRE/GR DBD interface (yellow); (middle) cortisol (magenta) inserted into the interface viewed from the major groove as in Fig. 4 and (bottom) cortisol inserted into the interface viewed from the minor groove.

Ligand insertion into DNA-receptor complexes

Docking of hormone into the wound complex The interface between the GR DBD and wound GRE is exposed and can be contacted by cortisol at 5'-TG-3'
5'-CA-3' [70]. No stereospeci®c hydrogen bonds were formed in the complex. The total energy of interaction of cortisol thus resulted from van der Waals contacts and was ÿ11.859 kcal broken down into ÿ9.375 kcal for the GRE and ÿ2.484 kcal for the GR DBD. The unnaturally occurring mirror image enantiomer of cortisol, ent-cortisol, was also capable of contacting the complex; hydrogen bonding was absent. The total energy was ÿ10.712 kcal due to van der Waals contacts with ÿ9.014 kcal for the GRE and ÿ1.698 kcal for the GR DBD. Docking of hormone into the unwound complex Cortisol was able to ®t remarkably well into the cavity in the unwound GRE/GR DBD complex (Figs 3, 4 and 5). The surface of the steroid and the cavity were complementary and six stereospeci®c hydrogen bonds were formed linking all functional groups on cortisol and heteroatoms in the protein/ DNA cavity. The orientation of the steroid in the complex was as previously reported with space ®lling models, i.e. the A/B ring stacked between bases C and A and the C/D ring stacked between T and G [66]. The C/D ring was closest to the GR DBD. The hydrogen bonds between the two carbonyl groups of cortisol and phosphate oxygens on adjacent strands as well as a water bridge between the 11b-hydroxyl and the O4 of T were also consistent with space ®lling models. Interestingly, a water molecule was also found at this location linked to T in the original crystal structure of the GRE/GR DBD [82]. The locations of the remaining hydrogen bonds in the ternary complex differed from those previously reported with DNA alone. The 17a hydroxyl linked better to the N7 of G in contrast to the O4' of deoxyribose attached to G shown by space ®lling models [66]. The 21 and 17a hydroxyls formed additional linkages to Lys 490 and Arg 466 in the GR DBD, respectively. The total van der Waals docking energy was ÿ26.163 kcal with ÿ20.934 kcal for the GRE and ÿ5.229 kcal for the GR DBD. The total electrostatic energy for hydrogen bonds was ÿ64.076 kcal with the locations and values for hydrogen bonds as follows: 3-CO . . .HOP (CpA), ÿ12.582 kcal; 20-CO . . . HOP (TpG), ÿ12.118 kcal; 11-HO . . .HOH . . .O4 (T), ÿ9.248 kcal; 17-OH . . .N7 (G), ÿ7.314 kcal; 17-HO . . .HN (Arg 466), ÿ9.875 kcal; 21-HO . . .HN (Lys 490), ÿ12.939 kcal. Total docking energy was ÿ90.239 kcal. In order to ®t ent-cortisol into the cavity, it was necessary to alter the conformation of Arg 466. Although not shown, poor complementarity was universally exhibited upon insertion of ent-cortisol into the cavity. The total van der Waals docking energy

83

was ÿ20.936 kcal with ÿ18.277 kcal for the GRE and ÿ2.659 kcal for the GR DBD. Poor overall electrostatic interactions totalling ÿ2.510 kcal were observed due to: the loss hydrogen bonding in the complex at Arg 466 measured at 17.247 kcal; the formation of only one viable hydrogen bond i.e. 21 HO . . .HN (Lys 490), ÿ12.964 kcal; relatively weak interactions at 3-CO . . .HOP (CpA), ÿ2.635 kcal and 20CO . . .HOP (TpG), ÿ4.158 kcal. The total docking energy was ÿ23.446 kcal.

DISCUSSION

Computer modeling demonstrates insertion of hormone into the cavity formed in the receptor DBD/unwound HRE interface Molecular modeling using both computer graphics and energy calculations demonstrates that the glucocorticoid hormone cortisol can contact the GRE in the presence of the GR DBD; unwinding of the GRE results in bending of the DNA concomitant with the formation of a cavity at the nucleic acid/protein interface into which cortisol ®ts in a complementary stereochemical manner. The van der Waals surface of the cortisol molecule conforms remarkably well to the surface of the cavity. Cortisol forms six stereospeci®c hydrogen bonds of which four link to DNA and two link to the receptor. The orientation of the steroid upon docking is as previously shown without receptor [66]. As summarized in Fig. 6, both the GR DBD and GRE contribute to a highly favorable energy change of approximately ÿ90 kcal upon insertion of the steroid. That these ®ndings are not coincidental is supported by the poor ®t of the unnaturally occurring mirror image of cortisol, ent-cortisol, which would be predicted not to exhibit complementarity. While ent-cortisol was capable of contacting the wound GRE/GR DBD interface with a similar energy of interaction as cortisol, it could not be inserted into the unwound GRE/DBD without destabilizing the site by disrupting hydrogen bonds between Arg 466 and the GRE. Even when forced into the unwound site, ent-cortisol was a poorer ®t than cortisol by more than 60 kcal. Forced insertion of ent-cortisol would impair unwinding and bending of DNA. These results demonstrate that the absolute chirality of the natural steroid is uniquely complementary to the chirality of the unwound GRE/GR DBD complex. Hormonal speci®city is inherent in the stereochemistry of the insertion site Since all ligands in the steroid/thyroid superfamily ®t between base pairs in the same sequence 5'-TG3'
5'-CA-3', other factors must govern the speci®city of the interaction [66, 67]. Because each hormone has unique donor/acceptor hydrogen bonds to DNA and is accommodated in cavities with different confor-

84

L. B. Hendry et al.

Fig. 6. The change in van der Waals, electrostatic and total energies resulting from insertion of cortisol and ent-cortisol into the wound and unwound GRE/GR DBD. The individual contributions of the GRE and GR DBD to the ®t of the steroids within the complex are also plotted.

mations including degrees of unwinding, we postulated that receptor proteins control the speci®city of insertion. This view is consistent with the ®nding that in the unwound GRE/GR DBD, Lys 490 facilitates hydrogen bonding of the 20 carbonyl group of cortisol to protonated phosphate. It is reasonable to expect that the differences in stereochemical features of other receptors will have distinct but equally important roles in governing the insertion of their cognate ligands. Stereospeci®c insertion should be assisted by other domains of the receptor including the LBD and amino terminal domain as well as other transcription factors. The details of such interactions await further structural elucidation of the interaction of full length receptors with HREs and should further elucidate differences in hydrogen bonding. Exposure and unwinding of the GRE at the insertion site In our examination of the wound GRE/GR DBD complex and construction of the unwound GRE/GR DBD complex, it became clear that much of the surface of the half site 5'-TGTTCT-3' which binds speci®cally to the receptor is covered by the protein. In fact, the major groove which would generally have the greatest exposure for contact with a candidate ligand is not easily accessible except for the ®rst two bases 5'-TG-3' and the complementary bases 5'-CA3' of the opposite strand. Only O6 and N7 of G form hydrogen bonds with Arg 466. It follows that unwinding of the GRE in the presence of the receptor would be limited to 5'-TG-3' leading to the formation of a cavity between these bases as shown in the model (Fig. 2). Moreover, the direction of unwinding would be constrained toward the GR DBD by the

double helical structure of the GRE. This results in bending of the DNA so that it conforms to the vshape of the GR DBD dimer. Interestingly, Arg 466 is part of a recognition helix which is conserved throughout the steroid/thyroid receptor superfamily and has been demonstrated to be generally important in DNA binding. In the case of the estrogen receptor (ER), NMR data demonstrate a hydrogen bonding linkage between the equivalent arginine of the ER DBD recognition helix and the equivalent G of the estrogen response element (ERE), 5'-TGACCT3' [86]. Thus, unwinding of the ERE in the presence of the ER DBD would also be initiated at 5'-TG-3' because the remaining bases are contacted by protein. These ®ndings suggest that the recognition helix including Arg 466 may serve as a general anchor for unwinding of HREs and that bending of DNA toward the receptor DBD may be a universal phenomenon throughout the superfamily. It follows that ligand insertion in the superfamily would be limited to the 5'-TG-3' of the HRE half site. Mutations in the insertion site which attenuate ®t of hormone diminish transcriptional activity elicited by hormone A particularly intriguing discovery is that of Hyder et al. demonstrating a striking homology between the full consensus estrogen response element, GGTCAnnnTGACC, and a thirteen base pair palindromic sequence in the 5' ¯anking region of the c-fos oncogene, GGTCTnnnAGACC [87]. In various transfection experiments, the latter sequence which does not contain the site that ®ts estradiol could not be induced by estradiol and was completely silent. However, a change in the ®rst half site to

Ligand insertion into DNA-receptor complexes

GGTCAnnnAGACC which provides a site to ®t estradiol conferred partial hormone responsiveness [88]. Of special signi®cance is the ®nding that estrogen responsiveness was also observed with GGTCGnnnAGACC. Unwinding of 5'-CG3'
5'-CG-3' results in a cavity which of the possible alternative sequences is the most closely related to 5'TG-3'
5'-CA-3' [66] (e.g. both are 5'-pyrimidine-3'purine sequences) and can also accommodate estradiol. Conversely, unwinding of the sequence 5'-CT3'
5'-AG-3' which is unresponsive to estradiol results in a very different cavity which is a relatively poor ®t for estradiol [66]. Similar ®ndings have been reported in the case of thyroid hormone [89]. A change in the end of a thyroid response element half site from CA which ®ts T3 to CT which is a poor ®t for T3 severely compromised T3-induced transcriptional activity. Factors which will affect ligand insertion The current working model is a `snapshot' of multiple, highly coordinated interactions among complex macromolecules. As additional information about the three dimensional structure of intact receptor proteins becomes available as well as computational techniques which can accurately model environmental factors such as the effects of solvent, micro pH, ionic strength, metal ions, conformational changes in the nucleic acid and protein, phosphorylated residues as well as other transcription factors, the current prototype will be re®ned. In this regard, recent studies using molecular dynamics calculations of solvated GRE/GR DBD without ligand demonstrate bending and unwinding of DNA [90] which are consistent with our model. Ligand insertion is also consistent with the early model of Duax and Weeks based on Xray crystallography and receptor binding studies which suggested that the steroid A ring would be attached to the receptor allowing for the D ring to interact with DNA [91]. The same laboratory raised the possibility that steroids might be potential intercalators [92]. The orientation of the steroid in our model which is based upon the constraints of insertion into the unwound DNA at the GRE/DBD interface is in agreement with their ®ndings. Folding of the receptor concomitant with transfer of the hormone to the DNA Taken as a whole, the weight of current evidence supports ligand insertion as a additional step in genomic hormone action. Such a mechanism (Fig. 1) would suggest that the relatively large LBD of the receptor folds upon itself and brings the ligand in proximity to the HRE [70]. We would expect ligand insertion into the cavity in the unwound DNA/DBD complex to be rapidly reversible; thus, it may also be feasible for the LBD to remove previously inserted hormone from the complex. Evidence for receptor folding has been reported for the androgen receptor

85

in which there is an interaction between the distal amino terminal and steroid binding domains [93]. It has also been postulated that in the case of the GR the hinge region which lies between the GR DBD and GR LBD may facilitate a close interaction between these domains [20]. Recently, X-ray structures for the LBDs of the retinoic acid RXR-a receptor [94], RAR-g receptor [95] and the thyroid hormone receptor [96] were reported. Considerable folding was found in each of the LBDs and the ligands for the RAR g and thyroid receptor were found buried in hydrophobic pockets within the LBD core. Given the dynamic, reversible binding of hormonal ligands to their receptors, we propose that a transfer of ligand can occur between the LBD and the unwound HRE/DBD complex. Such a transfer is consistent with the ®nding that binding of the ER to DNA is not dependent upon the presence of estrogen [47] and results in allosteric changes which increase dissociation of the hormone from the LBD [48]. Although in vivo and in vitro results differ due to varying conditions [49], there is general agreement with the hypothesis that conformational changes in the LBD are required for full transcriptional activity [47, 48] and that the role of hormones in transcription activation may be played after receptor binding to DNA [97]. The lack of effect of hormone on the binding of the GR to the GRE [50] despite well documented multifold increases in transcription upon treatment with ligand [20, 21] leads to the same conclusion. Pivotal role of Arg 466 which is conserved in the DBDs throughout the superfamily If ligand insertion proves to be a step in hormone action, the capacity to affect transcription would be ultimately dependent upon the formation of a ternary complex in which both the receptor and DNA play critical roles in the recognition of the ligand. Interference with any component of the process leading to insertion including conformational changes in the LBD would reduce hormonal activity. Ligand insertion would help explain why changes in the ®rst base T in the GRE result in loss of transcriptional activity [21] although this base is not contacted by the GR DBD [82]. It would also be consistent with the ®nding that the full length GR does interact with this base [50]. Mutations in any part of the receptor that affect the capacity to unwind the HRE or those critically involved in insertion or recognition of the ligand would have deleterious effects on transcriptional activity. While such mutations could be in any region of the receptor, the greatest effects on transcription would result from changes of those amino acids directly contacting the ligand and/or DNA. In our model, changes in the conserved arginine (Arg 466 of the GR) which is located in the recognition helix throughout the entire receptor superfamily and which

86

L. B. Hendry et al.

is crucial for formation of the unwound site would prevent hormone from acting. Mutagenesis experiments have indeed shown that alteration of Arg 466 in the GR results in the loss of transcriptional activity [98]. It is especially intriguing that mutations in this arginine have been found in other steroid receptors and have been linked to several diseases: vitamin D resistant rickets [99] and androgen insensitivity [100]. Hormonal activity correlates with ®t into the unwound HRE/DBD complex Loss of hormonal activity should also occur from changes in ligand structure which diminish ®t into the complex. For example, progesterone does not ®t as well into the unwound GRE/GR DBD as cortisol due to the absence of hydrogen bonding hydroxyl groups at the 17a, 11b and 21 positions and thus would be predicted by the model to have poor glucocorticoid activity. This ®nding is in agreement with the lack of glucocorticoid activity of progesterone even though under certain conditions it is equipotent to cortisol in binding to the GR [22]. We have recently shown that the glucocorticoid activity of various cortisol analogs correlates with ®t into the unwound GRE/GR DBD [101]. The action of certain antagonists could be due to the formation of complexes which prevent insertion of the natural hormone either by competing with hormone for binding to the receptor or to the unwound DNA/receptor complex. In the former case, the manner by which candidate antagonist structures bind to the receptor protein would be expected to result in different capacities to effect insertion and thus hormone function. This notion is consistent with differences observed in the location of antagonist binding sites in the estrogen receptor [42, 43] coupled with differences found in the conformations of the estrogen receptor resulting from the binding of different classes of antagonists [102]. Covalent binding of a ligand to the receptor at the hormone binding site would also prevent insertion and result in antagonist activity. This would explain the antiglucocorticoid activity of dexamethasone mesylate which links covalently to the glucocorticoid receptor[103]. Experimental approaches to further evaluate the ligand insertion hypothesis Further investigation of the current working model should help provide answers to a number of unresolved questions. What is the sequence of steps in ligand insertion; how does dimerization effect unwinding; can homo and heterodimers form two sites of unwinding; does unwinding take place in concert with DNA bending, etc.? Bending of DNA is now thought to be generally important in transcription (Ref. [78] and references therein). In the case of estrogens, bending occurs with the ER DBD and

increases with the full length receptor [73]. The addition of hormone has no apparent effect on bending. Similarly, estrogen is not required for receptor dimerization or DNA binding [47, 49]. Thus, the marked increases in transcription caused by estrogen [73] cannot be accounted for by binding of receptor to DNA, receptor dimerization or DNA bending and may be closely tied to the allosteric changes in the LBD which result in release of hormone [48]. In the case of glucocorticoids, hormone does not effect DNA binding yet increases dissociation of the GR from the GRE [50] raising the possibility that such allosteric changes may be acting in concert with ligand insertion. These results in the context of the generally poor relationship between receptor binding and hormonal activity again lead one to conclude that a second binding site must exist which recognizes the stereospeci®city and concentration of hormone [22]. While the argument can be made that other proteins could facilitate such recognition, the weight of current evidence including recent data demonstrating a correlation between degree of glucocorticoid activity and degree of ®t of cortisol analogs into unwound DNA [101] support the notion that this site is a relatively short lived ternary complex of receptor, unwound DNA and inserted ligand. It also follows that formation of the complex could be the rate limiting step in hormone action. Future experimental approaches to test this hypothesis will probably require stop/¯ow kinetic experiments in which rapidly reversible complexes can be measured. Correlation of differences in the DBDs and changes in cognate hormone structure suggests a ligand insertion domain in the receptor protein Work in progress comparing the structure of various DBDs has led us to speculate that a `ligand insertion domain' may exist which is unique to each receptor of the superfamily [104, 105]. This is based upon the ®nding that the extent of structural variations which occur in the natural hormones correlates with the degree of changes of the amino acid residues in their cognate DBDs. Moreover, the areas of least conservation are those residues in the dimerization interface and those along the surfaces of the DBD closest to the ligand insertion site which are adjacent to Arg 466. These amino acids would be expected to affect the extent of DNA unwinding and restrict insertion only to certain ligands. A putative noncontiguous ligand insertion domain in the full length receptor would help explain why certain mutations in the GR DBD do not diminish DNA binding yet abolish transcriptional activity [98]. A non-functioning ligand insertion domain might be found in certain orphan receptors which have no known ligand. A functional ligand insertion domain would facilitate bending and folding of the receptor thereby placing the ligand in the precise location for insertion. It will

Ligand insertion into DNA-receptor complexes

be of interest to see whether the putative ligand insertion domains coincide with transcription activation domains described by other workers [16]. CONCLUDING REMARKS

It has been twenty years since the insertion of steroid hormones into DNA modulated by chromosomal proteins was hypothesized to be involved in gene regulation [64]. This notion eventually led to the discovery of a speci®c site that best accommodated steroids and thyroid hormone and the prediction that this sequence would be crucial in the function of hormone responsive genes [65, 66]. That this sequence is conserved at the ends of HRE half sites in the steroid/ thyroid superfamily, can contact hormone in the presence of the receptor DBDs and unwind to form a stereospeci®c site which ®ts the hormone strongly suggests that ligand insertion is a critical step in genomic action throughout the superfamily. Such a step does not violate the basic tenets of the classical genomic mechanism of hormone action, provides the most direct way in which the hormone can activate the gene and explains early reports of the close structural relationships between nucleic acids and steroids [8]. It is also a salient example of the critical importance of stereochemical complementarity in biological processes as originally proposed by Pauling and Delbruck in 1940 [106]. AcknowledgementsÐWe wish to thank many collaborators who have assisted in the early development of the concepts and ®ndings which led to this study, in particular Francis H. Witham, Orville L. Chapman, Marion S. Hutson, Lillian K. Campbell, Mary L. Rosser and Jamie Steinsapir; recent modeling of ecdysone analogs was conducted by Jamie Ellis. We also thank Lewis Roach for valuable assistance in the preparation of the manuscript. Partial funding was provided by the Georgia Research Alliance.

REFERENCES 1. O'Malley B. W. and Schrader W. T., The receptors of steroid hormones. Sci. Am. 234 (1976) 32±43. 2. Munck A., The interfacial activity of steroid hormones and synthetic estrogens. Biochim. Biophys. Acta 24 (1957) 507± 514. 3. Munck A., Scott J. G. and Engel L. L., The interaction of steroid hormones and coenzyme components. Biochim. Biophys. Acta 26 (1957) 397±407. 4. Jensen E. V., Remembrance: Gregory Pincus-catalyst for early receptor studies. Endocrinology 131 (1992) 1581±1582. 5. Bush I. E. and Mahesh V. B., Metabolism of 11-oxygenated steroids-2,2-methyl steroids. Biochem. J. 71 (1959) 718±742. 6. Jensen E. V. and Jacobson H. I., Basic guides to the mechanism of estrogen action. Recent Prog. Horm. Res. 18 (1962) 387±414. 7. Clever U. and Karlson P., Induktion von puff-veranderungen in den speicheldrusenchromosomen von Chironomus tentans durch Ecdyson. Exp. Cell Res. 20 (1960) 623±627. 8. Huggins C. and Yang N. C., Induction and extinction of mammary cancer. Science 137 (1962) 257±262. 9. Jensen, E. V., Jacobson, H. I., Flesher, J. W., Saha, N. N., Gupta, G. N., Smith, S., Colucci, V., Shiplacoff, D., Neumann, H. G., DeSombre, E. R. and Jungblut, P. W., Estrogen receptors in target tissues. In Steroid Dynamics, eds.

10.

11. 12. 13.

14. 15.

16. 17. 18.

19. 20. 21. 22. 23. 24. 25.

26.

27. 28.

29. 30. 31.

87

G. Pincus, T. Nakao and J. F. Tait. Academic Press, New York, 1966, pp. 133±156. Jensen E. V., Suzuki T., Kawashima T., Stumpf W. E., Jungblut P. W. and DeSombre E. R., A two step mechanism for the interaction of estradiol with rat uterus. Proc. Natl. Acad. Sci. USA 59 (1968) 632±638. Jensen E. V. and DeSombre E. R., Mechanism of action of the female sex hormones. Annu. Rev. Biochem. 41 (1972) 203±230. Toft D. and Gorski J. A., A receptor molecule for estrogens: Isolation from the rat uterus and preliminary characterization. Proc. Natl. Acad. Sci. USA 55 (1966) 574±581. Gorski J. A., Toft D., Shyamala G., Smith D. and Notides A., Hormone receptors: Studies on the interaction of estrogens with rat uterus. Recent Prog. Horm. Res. 24 (1968) 45± 80. Smith D. F. and Toft D. O., Steroid receptors and their associated proteins. Mol. Endocrinol. 7 (1993) 4±11. Weigel, N. L., Receptor phosphorylation. In Mechanism of Steroid Hormone Regulation of Gene Transcription, eds. M. J. Tsai and B. W. O'Malley. R. G. Landes, Austin, 1994, pp. 93±110. Tsai M.-J. and O'Malley B. W., Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63 (1994) 451±486. Tsai, M. -J. and O'Malley, B. W. (eds.), Mechanism Of Steroid Hormone Regulation Of Gene Transcription. R. G. Landes, Austin, 1994. Katzenellenbogen J. A., O'Malley B. W. and Katzenellenbogen B. S., Tripartite steroid hormone receptor pharmacology: Interaction with multiple effector sites as a basis for the cell- and promoter-speci®c action of these hormones. Mol. Endocrinol. 10 (1996) 119±131. Evans R. M., The steroid and thyroid hormone receptor superfamily. Science 240 (1988) 889±895. Spanjaard R. A. and Chin W. W., Reconstitution of ligandmediated glucocorticoid receptor activity by trans-acting functional domains. Mol. Endocrinol. 7 (1993) 12±16. Nordeen S. K., Suh B. J., Kuhnel B. and Hutchison C. A., Structural determinants of a glucocorticoid receptor recognition element. Mol. Endocrinol. 4 (1990) 1866±1873. Teutsch G., Gailard-Moguilewsky M., Lemoine G., Nique F. and Philibert D., Design of ligands for the glucocorticoid and progestin receptors. Biochem. Soc. Trans. 19 (1991) 901±908. Brann D. W., Hendry L. B. and Mahesh V. B., Emerging diversities in the mechanism of action of steroid hormones. J. Steroid Biochem. Mol. Biol. 52 (1995) 113±133. Raynaud J.-P., Bouton M.-M., Gallet-Bourquin D., Philibert D., Tournemine C. and Azadian-Boulanger G., Comparative study of estrogen action. Mol. Pharmac. 9 (1973) 520±533. Raynaud, J. -P. and Ojasoo, T., The relevance of structure± af®nity relationships in the study of steroid hormone action. In Steroid Hormone Receptors: Structure and Function, eds. H. Eriksson and J. -A. Gustafsson. Elsevier Science Publishers, 1983, pp. 141±170. Raynaud, J. P., Ojasoo, T., Bouton, M. M., Bignon, E., Pons, M. and Crastes de Paulet, A., Structure±activity relationships of steroid estrogens. In Estrogens in the Environment, ed. J. A. McLachlan. Elsevier Science Publishing, 1985, pp. 24±42. Peters R. H., Crowe D. F., Avery M. A., Chong W. K. M. and Tanabe M., 11b-Nitrate estrane analogues: Potent estrogens. J. Med. Chem. 32 (1989) 2306±2310. Meyers C. Y., Kolb V. M. and Dandliker W. B., Doisynolic acids: Potent estrogens with very low af®nity for estrogen receptors. Res. Commun. Chem. Pathol. Pharmacol. 35 (1982) 165±168. Korach K. S., Chae K., Levy L. A., Duax W. L. and Sarver P. J., Diethylstilbestrol metabolites and analogs. J. Biol. Chem. 264 (1989) 5642±5647. Korach K. S., Chae K., Gibson M. and Curtis S., Estrogen receptor stereochemistry: Ligand binding and hormonal responsiveness. Steroids 56 (1991) 263±270. Kilbourn M. R., Arduengo A. J., Park J. T. and Katzenellenbogen J. A., Conformational analysis of nonsteroidal estrogens: The effect of conformer populations on the

88

32.

33. 34. 35.

36.

37.

38. 39. 40.

41. 42. 43.

44.

45.

46. 47.

48.

49.

50. 51.

L. B. Hendry et al. binding af®nity of meso- and DL-hexestrol to the estrogen receptor. Mol. Pharmac. 19 (1981) 388±398. Chernayaev G. A., Barkova T. I., Egorova V. V., Sorokina I. B., Ananchenko S. N., Mataradze G. D., Sokolova N. A. and Rozen V. B., A series of optical, structural and isomeric analogs of estradiol: A comparative study of the biological activity and af®nity to cytosol receptor of rabbit uterus. J. Steroid Biochem. 6 (1975) 1483±1488. Martucci C. and Fishman J., Uterine estrogen receptor binding of catecholestrogens and of estetrol (1,3,5(10)-estratriene3,15a,16,17a-tetrol). Steroids 27 (1976) 325±333. Zeelen F. J., Determination of the af®nity of a steroid for its receptor is not suf®cient to measure its intrinsic hormonal activity. Steroids 55 (1990) 325±326. van Aswegen C. H., Purdy R. H. and Wittliff J. L., Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers. J. Steroid Biochem. 32 (1989) 485±492. Pilat M. J., Hafner M. S., Kral L. G. and Brooks S. C., Differential induction of pS2 and cathepsin D mRNAs by structurally altered estrogens. Biochemistry 32 (1993) 7009± 7015. Brooks, S. C., Wappler, N. L., Corombos, J. D. and Doherty, L. M., Estrogen structure±receptor function relationships. In Recent Advances in Steroid Hormone Action, ed. V. K. Moudgil. Walter de Gruyter, New York, 1987, pp. 443±466. Jorgensen W. L., Rusting of the lock and key model for protein±ligand binding. Science 254 (1991) 954±955. Gabbard R. B., Hamer L. F. and Segaloff A., Structure±activity relationships of four 11-hydroxyestrones isomeric at the C-9 and C-11 positions. Steroids 37 (1981) 243±255. Wolff, M. E., Structure±activity relationships in glucocorticoids. In Glucocorticoid Hormone Action, eds. J. D. Baxter and G. G. Rousseau. Springer-Verlag, New York, 1979, pp. 100± 107. Ojasoo T., Raynaud J. P. and Dore J. C., Af®liations among steroid receptors as revealed by multivariate analysis of steroid binding data. J. Steroid Biochem. Mol. Biol. 48 (1994) 31±46. Hedden A., Muller V. and Jensen E. V., A new interpretation of antiestrogen action. Ann. N. Y. Acad. Sci. 761 (1995) 109±120. Martin P. M., Berthois Y. and Jensen E. V., Binding of antiestrogens exposes an occult antigenic determinant in the human estrogen receptor. Proc. Natl. Acad. Sci. USA 85 (1988) 2533±2537. Berthois, Y., Pons, M., Dussert, C., Crastes de Paulet, A. and Martin, P. -M., Agonist±antagonist activity of anti-estrogens in the human breast cancer cell line MCF-7: An hypothesis for the interaction with a site distinct from the estrogen binding site. Mol. Cell. Endocrinol., 1994, 259±268. Teutsch G., Nique F., Lemoine G., Bouchoux F., Cerede E., Gof¯o D. and Philibert D., General structure±activity correlations of antihormones. Ann. N. Y. Acad. Sci. 761 (1995) 5± 28. Suthers M. B., Pressley L. A. and Funder J. W., Glucocorticoid receptors: Evidence for a second non-glucocorticoid binding site. Endocrinology 99 (1976) 260±269. Murdoch F. E., Meier D. A., Furlow J. D., Grunwald K. A. A. and Gorski J., Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol. Biochemistry 29 (1990) 8377±8385. Fritsch M., Welch R. D., Murdoch F. E., Anderson I. and Gorski J., DNA allosterically modulates the steroid binding domain of the estrogen receptor. J. Biol. Chem. 267 (1992) 1823±1828. Zhuang Y., Katzenellenbogen B. S. and Shapiro D. J., Estrogen receptor mutants which do not bind 17b-estradiol dimerize and bind to the estrogen response element in vivo. Mol. Endocrinol. 9 (1995) 457±466. Truss M. and Beato M., Steroid hormone receptors: Interaction with deoxyribonucleic acid and transcription factors. Endocr. Rev. 14 (1993) 459±479. Kuiper G. G.J. M., Enmark E., Pelto-Huikko M., Nilsson S. and Gustafsson J.-A., Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. USA 93 (1996) 5925±5930.

52. Nutter L. M., Ngo E. O. and Abul-Hajj Y. J., Characterization of DNA damage induced by 3,4-estrone-oquinone in human cells. J. Biol. Chem. 266 (1991) 16380± 16386. 53. Telang N. T., Suto A., Wong G. Y., Osborne M. P. and Bradlow H. L., Induction by estrogen metabolite 16a-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells. J. Natl. Cancer Inst. 84 (1992) 634±638. 54. Liehr J. G., Gladek A., Macatee T., Randerath E. and Randerath K., DNA adduct formation in liver and kidney of male Syrian hamsters treated with estrogen and/or a-napthyl¯avone. Carcinogenesis 12 (1991) 385±389. 55. DeSombre E. R., Sha®i B., Hanson R. N., Kuivanen P. C. and Hughes A., Estrogen receptor-directed radiotoxicity with Auger electrons: Speci®city and mean lethal dose. Cancer Res. 52 (1992) 5752±5758. 56. Abul-Hajj Y. J., Tabakovic K. and Tabakovic I., An estrogennucleic acid adduct. Electroreductive intermolecular coupling of 3,4-estrone-o-quinone and adenine. J. Am. Chem. Soc. 117 (1995) 6144±6145. 57. Kuramochi H., Conformational studies and electronic structures of tamoxifen and toremifene and their allylic carbocations proposed as reactive intermediates leading to DNA adduct formation. J. Med. Chem. 39 (1996) 2877±2886. 58. Potter G. A., McCague R. and Jarman M., A mechanistic hypothesis for DNA adduct formation by tamoxifen following hepatic oxidative metabolism. Carcinogenesis 15 (1994) 439± 442. 59. Osborne M. R., Hewer A., Hardcastle I. R., Carmichael P. L. and Phillips D. H., Identi®cation of the major tamoxifendeoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res. 56 (1996) 66±71. 60. Hard G. C., Iatropoulos M. J., Jordan K., Radi L., Kaltenberg O. P., Imondi A. R. and Williams G. M., Major difference in the hepatocarcinogenicity and DNA adduct forming ability between toremifene and tamoxifen in female Crl:CD(BR) rats. Cancer Res. 53 (1993) 4534±4541. 61. Jarman M., Poon G. K., Rowlands M. G., Grimshaw R. M., Horton M. N., Potter G. A. and McCague R., The deuterium isotope effect for the a-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D5-ethyl]tamoxifen. Carcinogenesis 16 (1995) 683±688. 62. White I. N. H., de Matteis F., Davies A., Smith L. L., Crofton-Sleigh C., Venitt S., Hewer A. and Phillips D. H., Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells. Carcinogenesis 13 (1992) 2197±2203. 63. Pathak D. N. and Bodell W. J., DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems. Carcinogenesis 15 (1994) 529±532. 64. Hendry L. B., Witham F. H. and Chapman O. L., Gene regulation: The involvement of stereochemical recognition in DNA-small molecule interactions. Perspect. Biol. Med. 21 (1977) 120±130. 65. Hendry, L. B., Bransome, E. D., Jr., Hutson, M. S. and Campbell, L. K., U.S. Patent No. 4,461,619, 1984. 66. Hendry L. B., Stereochemical complementarity of DNA and steroid agonists and antagonists. J. Steroid Biochem. 31 (1988) 493±523. 67. Hendry L. B., Chu C. K., Rosser M. L., Copland J. A., Wood J. C. and Mahesh V. B., Design of novel antiestrogens. J. Steroid Biochem. Mol. Biol. 49 (1994) 269±280. 68. Hendry L. B., Drug design with a new type of molecular modeling based on stereochemical complementarity to gene structure. J. Clin. Pharmac. 33 (1993) 1173±1187. 69. Hendry, L. B., Design of estrogens and antiestrogens. In Receptor Research Methods, ed. B. Greenstein. Harwood, Amsterdam, 1995, pp. 279±318. 70. Hendry L. B. and Mahesh V. B., A putative step in steroid hormone action involves insertion of steroid ligands into DNA facilitated by receptor proteins. J. Steroid Biochem. Mol. Biol. 55 (1995) 173±183. 71. Lu X. P., Eberhardt N. L. and Pfahl M., DNA bending by retinoid X receptor-containing retinoid and thyroid hormone receptor complexes. Mol. Cell. Biol. 13 (1993) 6509±6519.

Ligand insertion into DNA-receptor complexes 72. Potthoff S. J., Romine L. E. and Nardulli A. M., Effects of wild type and mutant estrogen receptors on DNA ¯exibility, DNA bending and transcription activation. Mol. Endocrinol. 10 (1996) 1095±1106. 73. Nardulli A. M., Grobner C. and Cotter D., Estrogen receptor-induced DNA bending: Orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence. Mol. Endocrinol. 9 (1995) 1064± 1076. 74. Nardulli A. M. and Shapiro D. J., Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending. Mol. Cell. Biol. 12 (1992) 2037±2042. 75. Nardulli A. M., Greene G. L. and Shapiro D. J., Human estrogen receptor bound to an estrogen response element bends DNA. Mol. Endocrinol. 7 (1993) 331±340. 76. Carballo M. and Beato M., Binding of the glucocorticoid receptor induces a topological change in plasmids containing the hormone-responsive element of mouse mammary tumor virus. DNA Cell Biol. 9 (1990) 519±525. 77. Leidig F., Shepard A. R., Zhang W., Stelter A., Cattini P. A., Baxter J. D. and Eberhardt N. L., Thyroid hormone responsiveness in human growth hormone-related genes. J. Biol. Chem. 267 (1992) 913±921. 78. Kim J., Klooster S. and Shapiro D. J., Intrinsically bent DNA in a eukaryotic transcription factor recognition sequence potentiates transcription activation. J. Biol. Chem. 270 (1995) 1282±1288. 79. King I. N., de Soyza T., Catanzaro D. F. and Lavin T. N., Thyroid hormone receptor-induced bending of speci®c DNA sequences is modi®ed by an accessory factor. J. Biol. Chem. 268 (1993) 495±501. 80. Pendergast P., Pan Z. and Edwards D. P., Progesterone receptor-induced bending of its target DNA: Distinct effects of the A and B receptor forms. Mol. Endocrinol. 10 (1996) 393±407. 81. Ribeiro R. C. J., Kushner P. J. and Baxter J. D., The nuclear hormone receptor gene superfamily. Annu. Rev. Med. 46 (1995) 443±453. 82. Luisi B. F., Xu W. X., Otwinowski Z., Freedman L. P., Yamamoto K. R. and Sigler P. B., Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352 (1991) 497±505. 83. Baumann H., Paulsen K., Kovacs H., Berglund H., Wright A. P. H., Gustafsson J.-A. and Hard T., Re®ned solution structure of the glucocorticoid receptor DNA-binding domain. Biochemistry 32 (1993) 13463±13471. 84. Bernstein F. C., Koetzle T. F., Williams G. J. B., Meyer E. F. Jr., Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T. and Tasumi M., The protein data bank: A computerbased archival ®le for macromolecular structures. J. Mol. Biol. 112 (1977) 535±542. 85. Abola, E. E., Bernstein, F. C., Bryant, S. H., Koetzle, T. F. and Weng, J., Protein data bank in crystallographic databases: Information content, software systems, scienti®c applications. In Crystallographic Databases: Information Content, Software Systems, Scienti®c Applications, eds. F. H. Allen, G. Bergerhoff and R. Sievers. Data Commission of the International Union of Crystallography, Bonn/Cambridge/Chester, 1987, pp. 107± 132. 86. Schwabe J. W. R., Chapman L., Finch J. T. and Rhodes D., The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: How receptors discriminate between their response elements. Cell 75 (1993) 567±578. 87. Hyder S. M., Stancel G. M. and Loose-Mitchell D. S., Presence of an estradiol response region in the mouse c-fos oncogene. Steroids 56 (1991) 498±504. 88. Nawaz Z., Stancel G. M., McDonnell D. P. and Hyder S. M., Creation of an active estrogen-responsive element by a single base change in the ¯anking sequence of a cellular oncogene: A possible mechanism for hormonal carcinogenesis?. Mol. Carcinogenesis 7 (1993) 76±82. 89. Brent G. A., Williams G. R., Harney J. W., Forman B. M., Samuels H. H., Moore D. D. and Larsen P. R., Capacity for cooperative binding of thyroid hormone (T3) receptor dimers de®nes wild type T3 response elements. Mol. Endocrinol. 6 (1992) 502±514.

89

90. Harris L. F., Sullivan M. R., Popken-Harris P. D. and Hickock D. F., Molecular dynamics simulations in solvent of the glucocorticoid receptor protein in complex with a glucocorticoid response element DNA sequence. J. Biomol. Struct. Dynamics 12 (1994) 249±270. 91. Duax, W. L. and Weeks, C. M., Molecular basis of estrogenicity: X-ray crystallographic studies. In Estrogens in the Environment, ed. J. A. McLachlan. Elsevier, North Holland, 1980, pp. 11±31. 92. Weeks C. M., Rohrer D. C. and Duax W. L., Deoxycorticosterone±adenine interactions in a crystalline complex. Science 190 (1975) 1096±1097. 93. Zhou Z., Lane M. V., Kemppainen J. A., French F. S. and Wilson E. M., Speci®city of ligand-dependent androgen receptor stabilization: Receptor domain interactions in¯uence ligand dissociation and receptor stability. Mol. Endocrinol. 9 (1995) 208±218. 94. Bourguet W., Ruff M., Chambon P., Gronemeyer H. and Moras D., Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-a. Nature 375 (1995) 377± 382. 95. Renaud J.-P., Rochel N., Ruff M., Vivat V., Chambon P., Gronemeyer H. and Moras D., Crystal structure of the RARg ligand-binding domain bound to all-trans retinoic acid. Nature 378 (1995) 681±689. 96. Wagner R. L., Apriletti J. W., McGrath M. E., West B. L., Baxter J. D. and Fletterick R. J., A structural role for hormone in the thyroid receptor. Nature 378 (1995) 690±697. 97. Beekman J. M., Allan G. F., Tsai S. Y., Tsai M.-J. and O'Malley B. W., Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain. Mol. Endocrinol. 7 (1993) 1266±1274. 98. Schena M., Freedman L. P. and Yamamoto K. R., Mutations in the glucocorticoid receptor zinc ®nger region that distinguish interdigitated DNA binding and transcriptional enhancement activities. Genes Dev. 3 (1989) 1590±1601. 99. Sone, T., Kerner, S. A., Saijo, T., Takeda, E. and Pike, J. W., Mutations in the DNA binding domain of the vitamin D receptor associated with hereditary resistance of 1,25-dihydroxyvitamin D3 In Vitamin D: Gene Regulation, Structure± Function Analysis and Clinical Applications, eds. A. W. Norman, R. Bouillon and M. Thomasset. Walter de Gruyter, New York, 1991, pp. 84±85. 100. Quigley C. A., De Bellis A., Marschke K. B., El-Awady M. K., Wilson E. M. and French F. S., Androgen receptor defects: Historical, clinical, and molecular perspectives. Endocrinol. Rev. 16 (1995) 271±321. 101. Hendry, L. B., Bransome, E. D., Jr. and Mahesh, V. B., A rate limiting step in the action of hormones in the steroid/ thyroid superfamily may involve formation of a ternary complex of receptor, unwound DNA and inserted ligand. 10th Int. Congr. Endocrinol., 1996, 135 (abstr. P1±2). 102. McDonnell D. P., Clemm D. L., Hermann T., Goldman M. E. and Pike J. W., Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol. Endocrinol. 9 (1995) 659±669. 103. Simons S. S. Jr., Pumphrey J. G., Rudikoff S. and Eisen H. J., Identi®cation of cysteine 656 as the amino acid of hepatoma tissue culture cell glucocorticoid receptors that is covalently labeled by dexamethasone 21-mesylate. J. Biol. Chem. 262 (1987) 9676±9680. 104. Mahesh, V. B., Bransome, E. D., Jr. and Hendry, L. B., Quantitative correlations between structural differences in receptor DNA binding domains, unwound hormone response elements and hormones are explained by a ligand insertion step in steroid hormone action. 10th Int. Congr. Endocrinol., 1996, 136 (abstr. P1±5). 105. Bransome, E. D., Jr., Mahesh, V. B. and Hendry, L. B., A ligand insertion domain in receptors in the steroid/thyroid superfamily is suggested by ternary complexes of receptor, hormone and unwound DNA. 10th Int. Congr. Endocrinol., 1996, 136 (abstr. P1±6). 106. Pauling L. and Delbruck M., The nature of the intermolecular forces operative in biological processes. Science 92 (1940) 77±79.