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Steroid structure and function. Molecular conformation of 4-hydroxyestradiol and its relation to other catechol estrogens
0022-473 l/ES $3.00 + 0.00 Copyright @ 1988 Pergamon Press pie
J. steroid Bfodwm. Vol. 29, No. 4, pp. 387-392, 1988 Primal in Great Britain. AI1rights mcrved
STEROID STRUCTURE AND FUNCTION. MOLECULAR NONFORMATION OF 4-HYDRO~EST~DIOL AND ITS RELATION TO OTHER CATECHOL ESTROGENS ZDZISLAWWAwRzAKt, WILLIAM L. DUAX$, PHYLLISD. STRONOand JUDITH Wnxaz* Medical Foundation of Buffalo, Inc., 73 High Street, Buffalo, NY 14203and *Departments of Obstetrics and Gynecology and Pharmacology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, U.S.A. (Received 2 July 1987)
~~-Hydroxylation of estrogens at C(2) or C(4) effects ~fferent~By their binding afEnity to and dissociation rate from the estrogen receptor. The X-ray crystal structure of 4-hydroxyestradiol (COH-Er) is reported here and compared with that of 2-hydroxyestradiol (2-OH-Er), the 2- and ehydroxylated derivatives of estrone (E, ) and with that of the parent estrogens, El and Es. The overall molecular shape and hydrogen bonding patterns of each were examined for their possible relevance to their binding to the estrogen receptor and their biological activity. A shift in the B-ring conformation away from the symmetrical 7a,8/?-half-chair form toward the @-sofa form is induced by both 2- and Chydroxy substitution. This shift appears to be larger in the case of Es than Et derivatives and to be correlated with an observed change in the hydrogen bonding potential of the C(3) hydroxyl. In 4-OH-Er, as in Er and 4-OH-E,, the C(3) hydroxyl functions both as a hydrogen bond donor and acceptor. In contrast in 2-OH-E, the hydroxyl functions only as a donor. The markedly reduced afhnity of 2-hydroxylated estrogens for the estrogen receptor could be due to a combination of steric interactions, competition between O(2) and O(3) for hydrogen bonds for a common site on the receptor, and to general interference with hydrogen bond formation of O(3). The C(4) hydroxyl participates in the formation of a chain of hydrogen bonds in the solid state that is similar to a chain seen in single crystals of Er. The presence of a similar chain of hydrogen bonds involving O(3) in the receptor site could account for the decreased dissociation rate of the 4-OH-E, receptor complex.
~ODU~ION
Catecholestrogens are formed by hydroxylation of the estrogen molecule at either the C(2) or the C(4) position. In the body as a whole 2-hydroxyla~on predominates and represents a major route of metabolism of phenolic estrogens [ 11. Microsomal cytochrome P-45O-dependent monooxygenase(s) which catalyzes the reaction and acts primarily as a 2-hydroxylase has been identified in liver as well as in several steroidogenic and estrogen target organs [l-6]. However, hydroxylation at the C(4) position also occurs [2,7J and may be prominent in certain targets of estrogen action. Thus anterior pituitary and brain tissue have been reported to convert estradiol in uitro to its 2- and 4-hydroxylat~ metabolites in a 1: 1 ratio [I, 7’J. In addition, it has been recently demonstrated that catecholestrogen formation may be mediated by certain peroxidases and by microsomes, by an organic hydroperoxidedependent peroxidatic mechanism that also generates the 2- and 4-hydroxylated metabolites in a 1: 1 ratio [8,9]. tOn a leave from Technical University of Lodz, Lodz, Poland. $To whom correspondence should be addressed. 381
The physiolo~cal role of catecholestrogens formed in estrogen target tissues is not known. However, they have been proposed to act as autocrine or paracrine mediators of some of the ef&cts of the parent hormone. The addition of a second hydroxyl group to ring A of phenolic estrogens markedly alters their biochemical characteristics. By virtue of their catechol structure they can inhibit cateehol-Omethyltransferase and tyrosine hydroxylase and, thereby, potentially influence the rate of biosynthesis or degradation of catecholamines [I, lo]. Acting in this capacity the Z- and Chydroxylated estrogens appear equipotent [7]. Substitution of a 2- or ehydroxyl group also changes the interaction of the estrogen molecule with its receptor. However, in this respect it is possible to differentiate between the consequences of 2- and 4-hydroxylation. While both substitutions reduce the affinity of the steroid for the receptor this effect is greater for 2- than Chydroxylation [l 11. Moreover, 2- and Chydroxylation have opposite effects on the dissociation rate of the estrogen-receptor complex. Hydroxylation of C(2) substantially increases while that at C(4) significantly decreases the dissociation rate of the steroid. Consequently, 2-hydroxylated estrogens are weak estrogen agonists whereas the ~hyd~xylat~
ZDZISLAW WAWRZAK er al.
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estrogens are potent and possibly long-acting estrogens. We have proposed that the steroid A-ring plays a critical role in initiating estrogen receptor binding and that the D-ring controls estrogen potencyjl21. The strength and orientation of hydrogen bonds involving O(3) is a dominant factor governing A-ring interaction. The X-ray crystallographic studies of 2and 4-hydroxy estrogens have been undertaken in order to determine the influence that these substitutions have upon the overall shape of the molecule and the strength and orientation of hydrogen bonds involving O(3). These studies were carried out in an effort to identify characteristic patterns in molecular conformation and hydrogen bonding interactions. The existence of such patterns would indicate an energetic preference or stability that would suggest the pattern of interactions found in the receptor site. We report here the results of the X-ray crystal structure analysis of 4-hydroxyestradiol (4-OH-Ez) together with a comprehensive analysis of the previously published structures of 2-OH-E2, 2-OH-E,, 4-OH-E, [13] and the parent compounds, E, and E, 1141.
EXPERIMENTAL
Crystals of 4-OH-E, were grown by slow evaporation from ethanol. Unit-cell parameters were determined by least-squares fit of the 8 values of 25 reflections. Intensity data were collected to f3 = 75” on an Enraf-Nonius CAD4 diffractometer using CuICcr radiation. The structure was solved by means of direct methods using the program MULTAN [ 151 and refined by full-matrix least-squares. All hydrogen atom positions were found from difference Fourier maps and refined with thermal parameters equal to those from corresponding nonhydrogen atoms. Nonhydrogen atoms were refined anisotropically. The crystal and refinement data are given in Table 1. The nonhydrogen atom coordinates and equivalent isotropic thermal parameters are collected in Table 2. Structure factors, atomic coordinates for the hydrogen atoms, anisotropic thermal parameters for nonhydrogen atoms and bond distances, bond angles and torsion angles for nonhydrogen atoms are available from the authors upon request.
Table
I. Crystal
and refinement
Molecular formula Molecular weight Crystal symmetry Space group Z Cell dimensions a, A
data
C,sH,,O, 288.4 Monoclinic p2, 2 7.0226(8) 19.2395(20) 5.7348(6) 107.806(2) 737.7(3)
Reliability factors Observed data All data
0.045/ I527 0.04611563
Table 2. Fractional atomic coordinates (x 104) and equivalent isotropic thermal parameters .&’ (x IO*) for nonhydrogen atoms with estimated standard deviations in parantheses Atom
x/a(c)
C(1) C(2)
462 l(4) 5108(4) 3662(4) 1748(4) 1246(3) - 836(4) - 1272(4) 548(3) 2238(4) 2729(4) 4074(4) 3550(4) 1944(3) 102(3) - 1541(4) - 1099(4) 951(4) 2760(5) 3916(3) 279(3) 2152(3)
C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(l0) C(l1) C(12) C(J3) C(14) C(15) C(16) C(17) C(l8) O(3) O(4) O(l7B)
y/b@ ) 76~2) 7104(2) 689 l(2) 7177(2) 7677(2) 7980(l) 8474(2) 8928 8455(2) 790 l(2) X867(2) 9453(2) 993 l(2) 9487(2) 10020(2) 10612(2) 10435(2) 10341(2) 6402(2) 6941(2) 11034(2)
z/c(a) 9661(5) 8 176(6) 6055(5) 5502(4) 6946(4) 6124(5) 7973(6) 9 190(4) 10721(4) 9101(4) 12231(6) 13740(5) 12 153(4) 10824(4) 97 1O(6) 11636(6) 13509(4) 10371(6) 4468(4) 3413(4) 14439f4)
B,(a) 328(7) 342(7) 302(6) 284(6) 266(6) 306(6) 353(7) 251(5) 277(6) 269(6) 385(7) 365(7) 249(5) 275(6) 388(8) 405(S) 285(6) 353(7) 400(6) 374(5) 357(5)
RESULTS
The overall geometry of the 4-OH-E2 molecule is shown in Fig. 1. When the geometry of 4-OH-E2 is compared with that of 2-OH-E2 [8], the bond lengths and angles are found to be in very good agreement. However, there is a small but significant difference in B-ring torsion angles and overall molecular shape. The distribution of conformations of the B-ring in estradiol, estrone, and their catechol derivatives [ 131, as a function of this ring symmetry is shown in Fig. 2. Generally, the B-ring conformation of 1,3,5(10)estratriene steroids vary from a 7ol,8/?-half-chair conformation in which atoms C(9), C( lo), C(5) and C(6) are coplanar and atoms C(7) and C(8) are equidistant from that plane on opposite sides (i.e. asymmetry
Fig. 1. A stereoscopic view of the 4-OH-E, molecule prepared using the program ORTEP. A stereo viewing device will permit the reader to see the molecule in three dimensions. Contact the authors for further information.
Catechol estrogen conformation and binding
70,BP
1 hoif
25 20 s
clwrir
i
p
15
‘V
h
u” Q
infmmtdiafe 5
10 i
I
0
5
10
d
15 20 25 AC2 (5-10)
30
Fig. 2. Relation of asymmetry parameters of the B-rings in 1,3,5~lO)~~at~ene structures. The rotational assets parameter AC@-IO) is plotted vs the mirror beets parameter AC*(S).E,(A), ZOH-E, (A), 4-OH-E, (V), E2 tO),2-OH-E, (0). 4-OH-& (W.
parameter 4<=,(5-lo)<7 [ill) to a 8/S-sofa conformation in which atoms C(9), C(lO), C(S), C(6) and C(7) are coplanar and C(8) is on the fi side of that plane (i.e. ~~rn~t~ parameter AC,(S)< 10 [16]). X-Ray diffraction studies of three crystal forms of estrone have been reported including one form containing two independent molecules in the crystallographic unit cell [14]. The conformation of estrone itself in three of the four observations (solid triangles in Fig. 2) is close to the symmetric 7a,8j?-half-chair form. The fourth conformer of estrone is si~ifi~ntly different and more nearly approximates the 8jS-sofa conformation. It is worth noting that the two conformers exhibiting the maximum difference are the pair that occur together in one crystal form. The 2- and Cbydroxy derivatives of estrone also crystallize with two independent molecules in the crystallographic unit cell. All derivatives of estrone have conformations lying midway betweeti these extremes with the 2-bydroxy derivatives being closest to the 8#?-sofa coronation. Tbe B-ring of estradiol determined in three independent observations (solid circles in Fig. 2) has a slightly distorted 7a,8J-half-chair conformation and *Average values for these angles in the nine structures in Fig. 2 having the 7a,8@-half-chair conformation (AC,(S)> 10.0 or AC+10)~ 10.0).
389
exhibits little variation. In contrast, the B-ring of 2-OH-E, (open circle) has the rarely observed perfect 8/I-sofa conformation. The B-ring in 4-OH-E2 (half circle) has a slightly distorted O/?-sofa conformation, i.e. shifted toward an intermediate 8/3-sofa-7a,8Bhalf-chair conformation. This shift in conformation toward the 8/l-sofa form with hydroxylation is apparently the result of the combined influence of the 17@and the 2- or Cbydroxy substitutions. The b-face interactions between the C(18) methyl group and the 17-hydroxy on one side and the axial bydrogens at C(8) and C(l1) on the other appear to cause the observed closing of the C(14)-C(8)-C(9)-C(ll) and C(S)-C(9)-C!(ll~(l2) torsion angles from 54 and 53’* to 51 and 50”, respectively. This twist about the C(8)-&(9) bond opens the C(7)-C(S)-C{9)-C( 10) torsion angle from 54” to 59” and shifts the B-ring toward the 8/3-sofa conformation. A preference for the 7a,8/?-half-chair conformation is illustrated in Fig. 3 for estradiol, estrone and the catechol derivatives of estrone. The observation of an 88-sofa conformation in only one of four crystallographically independent estrone structures suggests that, while this conformation is energetically feasible, it is less favored. Hydroxyl substitution in the 2- and 4-positions of estradiol appear to stabilize the 88-sofa conformation. The conformation of 4-OH-E, is intermediate between those of 2-OH-E, and estradiol (Fig. 3). Although the difference in conformation is subtle it may influence the interactive properties of the steroid surface or of the 3-bydroxyl group. Tbe angles around the C(3) of 4-bydroxyestradiol exhibit the characteristic distortion noted previously in 2-bydroxyestradiol, 4-bydroxyestrone and 2-hydroxyestrone[13]. In all four cases the largest angle is opposite the bond that is tram to the bydroxyl hydrogen [13]. In 4-OH-E, the two angles C(2j-C(3)-0(3) and C(4)-C(3)-0(3) differ by almost IO”. Tbe angles around the C(4) atom in 4-bydroxyestradiol do not follow this pattern. Tbe C(3)-C(4)O(4) and C(5~(4~(4) angle differ by less tban 1” and the distortion is in the opposite direction. The angle opposite the bond that is trans to the bydroxyl hydrogen is the smaller of the two. Tbe bydroxyl hydrogens are almost coplanar with the A-ring in 4-OH-E, where the torsion angles defining deviations from coplanarity are -2” for C(3)474)-0(4)-H(40) and 6” for C(2)-C(3)--0(3)-H(30). Values for
Fig. 3. Stereo view (see caption to Fig. I) of a least-squares fit of & (solid Ike) against 4-OH-E, and 2-OH-E, (dashed lines). The least-squares process minimized the separation between corresponding atoms in the C- and D-rings of the two structures.
ZDZISLAW WAWRZAK et al.
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O(
Fig. 4. Hydrogen bond geometry of the A-ring of (a) 4-OH-E,, (b) &, (c) 4-OH-E, and (d) 2-OH-E,. Dashed lines (---) represent intermolecular hydrogen bonds and dot dashed lines (-.--) represent intramolecular hydrogen bonds. Acceptor and donor atoms on adjacent molecules include water and 0(17) oxygens. Note that the O(3) hydroxyl acts as a hydrogen bond donor and acceptor in (a), (b) and (c) but as a bifurcated donor only in (d).
2-OH-E2 are - 18” for C(lw(2)-0(2kH(20) and 5” for C(2)-C(3)-0(3)-H(30) [13]. The O(3) atom of 4-OH-E, accepts an intramolecular hydrogen bond from O(4) and donates a hydrogen bond to the 0( 17) of an adjacent molecule. The O(4) atom of 4-OH-E* accepts a hydrogen from the 0(17) of another nearby steroid (Fig. 4a). This hydrogen bonding pattern is compared with those observed in crystals of E,, 4-OH-E,, and 2-OH-E, in Fig. 4. DISCUSSION
X-Ray analysis reveals that 2- and 4-hydroxylation of estradiol and estrone induces subtle alterations in the overall shape of the steroid and much more obvious alterations of the hydrogen bonding capabilities of the C(3)-hydroxyl. The changes associated with the different substitutions offer insight into the molecular basis for the variation in binding and activity of the 2- and 4-hydroxy derivatives. Relatively poor binding of the 2-hydroxy derivatives of estrone and estradiol, despite almost identical conformations of 2-hydroxy- and 4-hydroxyestradiol,
suggest the O(2) hydroxyl may be directly responsible for the decreased binding affinity. There are two possible ways in which a 2-hydroxy substituent could interfere with receptor binding. Either the 2-hydroxy substituent would penetrate the receptor surface when the steroid is properly oriented or the 2-hydroxy would mimic the 3-hydroxy in its association with the receptor and generate a misorientation of the ligand. In the first case the 2-hydroxy would constitute a steric hindrance to binding and in the second case it might promote inappropriate and nonproductive binding. An association of the steroid with the receptor in which the 2-hydroxy substituent in 2-OH-E, mimicked the 3-hydroxy in E, (Fig. 5) would lack the proper orientation of the 17B-hydroxy substituent and contribute to the observed enhancement in the rate of dissociation. Both of these interpretations would be consistent with increased dissociation rates of the receptor complexes of 2-OH-E, and 2-OH-E*, compared to their parent steroid. In contrast to the effect of a 2-OH, the presence of a 4-OH substituent appears to be well tolerated by the receptor. Furthermore, the hydrogen bond pattern established between 4-OH-E, and the receptor
Catechol estrogen conformation
Fig. 5. View of a least-squares superposition of Es (solid line) and 2-OH-E, (dashed line) in which the Z-OH substituent mimics the 3-hydroxy group of estradiol. may strengthen the association and decrease the dissociation rate. Two interrelated aspects of the hydrogen bonding pattern observed in the solid state support this contention, the numbers of individualhydrogen bonds formed and their presumed strengths as reflected in their length and geometry. In 5 out of 7 estrone and estradiol structures the O(3) forms a strong hydrogen bond directing its hydrogen syn-periplanar to the C(2)-C(3) bond. The O(3) of estradiol always acts as an acceptor as well as a donor, whereas the O(3) of estrone acts as a donor only. Similarly, in 2-OH-E2 and 2-OH-E, the O(3) acts as a donor only and accepts no inter- or intramolecular hydrogen bonds. This could explain the lower dissociation rate of Ez relative to E, and 2-O&E,. In E, and E, the length of the hydrogen bonds in which O(3) acts as a donor ranges from 2.64 to 2.83A. In 2-OH-E, and 2-OH-E, the hydrogen bond lengths are significantly larger (2.93 and 3.08 A) and presumably weaker than the comparable bonds in estrone and estradiol. This observation is consistent with the lower affinity of the 2-hydroxy derivatives of E, and E, relative to their parent compounds. In contrast, in 4-OH-E, and both c~stallo~aphi~lly independent 4-0X-I-E, molecules the O(3) acts as an intramolecular hydrogen bond acceptor as well as an intermolecular hydrogen bond donor. Furthermore in 4-OH-E, [13], the 4-hydroxy acts as an inter-
and binding
391
molecular hydrogen bond acceptor as well. There is theoretical [ 17j and experimental evidence [ 181 that the formation of a chain of three hydrogen bonds such as this enhances the strength of these hydrogen bonds. The lengths (2.73, 2.74 and 2.75& and, presumably, strengths of the hydrogen bonds formed in the crystal structures of 4-OH-E, and 4-OH-E, are similar to those observed in estradiol and estrone. Increased stability due to the formation of more, shorter, and stronger hydrogen bonds and their compatibility with the apparent location of hydrogen bond acceptor and donor sites on the receptor may account for the higher binding affinity and the slower dissociation rate from the receptor of 4-OH-E, and 4-OH-E,. The data available on binding affinities and dissociation rates as well as the hydrogen bonding characteristics of estrone, estradiol and their 2and ~hydroxylated derivatives are surnrna~~ in Table 3. It should be noted that while the addition of a Chydroxy group to estrone permits the O(3) to act as an acceptor as well as a donor, the addition of a 2-hydroxy group to estradiol results in a loss of that ability. The behavior observed in the solid state is consistent with the effect of these substitutions on the binding affinity of these compounds. Acknowledgements-This work was supported in part by NIAAMDD Grant No. AM-26546 and_DDR Grant Nd.
RR-05716 fto WLD’I and bv NICHHD Grant HD-09743 (to JW). T&e authors would*like to thank G. Del Rel, M.
Tugac and J. Gallmeyer for their technical assistance. The organization and analysis of the data base associated with the investigation, and some of the illustrations were carried out using the PROPHET system, a unique national computer resource sponsored by the NIH.
REFERENCES
Ball P. and Knuppen R.: Catecholestrogens (2- and 4-hydroxy estrogens). Chemistry, biogenesis, metabolism, occurrences and physiological significance. Actu. endoer., Copenkt. 93 (1980) l-127. Hersey R. M., Williams K. I. and Weisx J.: Catechol estrogen formation by brain tissue: Characterization of a direct product isolation assay for estrogen 2- and 4-hydroxylase activity and its application to studies of 2. and 4-hydroxyestradiol formation by rabbit hypothalamus. Endocrinology 109 (1981) 1912-1920.
TabIc 3. Hydrogen bond geometry, binding affinity sod dissociation rate of estradiol (E.& cstrone (E, f and their 2-hvdroxv and 4-hvdroxv derivatives
-
Hydrogen bond geometry 0(3)D,. . A(&* 0(3)A.. . L&h)*
4.OH-E, E, 2-OH-E, 4-OH-E,
2.70(3) 2.86(2) 2.73 2.80(4) 2.82(3) 2.73
2.76(2) 2.65 2.80
Estrogen receptor binding Dissociation? fi (min) RBA ailinity 100 24 45 11 2 11
95.0 11.3 130.0 8.7 3.7 15.1
*Hydrogen bond distances represent average values with number of contributors in parentheses. D and A refer to donor and aeocptor atoms in the hydrogen bond. tData from ref. 1I, rate of dissociation at 25°C.
392
ZDZISLAWWAWRWK et al.
3. Hoffman A. R., Paul S. M. and Axelrod J.: Catecholestrogen synthesis and metabolism by human breast tumors in vitro. Cancer Res. 39 (1979) 45844587. 4. Mondschein J. !I., Hersey R. M., Dey S. K., Davis D. L. and Weisz J.: Catechol estrogen formation by the pig blastocyst during the preimplantation period. Biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity. Endocrinology 117 (1985) 2339-2346. 5. Hammond J. M., Hersey R. M., Walgoa M. A. and Weisz J.: Catecholestrogen production by porcine ovarian cells. Endocrinology 118 (1986) 2292-2299. 6. Fishman J. and Norton B.:Catecholestrogen formation in the central nervous system of the rat. &docrinology 69 (1985) 1054-1059. I. Ball P., Haupt M. and Knuppen R.: Biogenesis and metabolism of catecholestrogens in vitro. In Catechol Estrogens (Edited by G. R. Merriam and M. Lipsett). Raven Press, New York (1983) 91-104. 8. Mondschein J. S., Hersey R. M. and Weisz J.: Purification and characterization of estrogen-2/ 4-hvdroxvlase activitv from rabbit hvnothalmi: per&da&z-mediated -&echo1 estrogen formation. EndocrinoloPv 119 (1986) 1105-l 112. 9. Weisz J. a;d Bui‘ Q.: ‘Multiple pathways of catecholestrogen formation: Characterization of NADPHand organic hydroperoxide-dependent 2- and 4-hydroxylation of estradiol by microsomes from human placenta and rat brain. 69rh Annual Meeting of the Endocrine Society, Indianapolis, Indiana, June 1987. Abstr. No. 782. 10. Lloyd T. and Weisz J.: Direct inhibition of tyrosine hydroxylase activity by catecholestrogen. J. biol. Chem. 253 (1978) 4841-4843.
11. MacLusky N. J., Bamea E. R., Clark C. R. and Naftolin E.: Catechol estrogens and estrogen receptors. In Catechol Estrogens (Edited by G. R. Merriam and M. B. Lipsett). Raven Press, New York (1983) pp. 151-165. 12. Duax W. L. and Weeks C. M.: Molecular basis of estrogenicity: X-Ray crystallographic studies. In Estrogens in the Environment (Edited by J. W. McLachlan). Elsevier North-Holland, New York (1980) pp. 11-31. 13. Daux W. L., Griffin J. F., Swenson D. C., Strong P. D. and Weisz J.: Steroid structure and function-IX. Molecular conformation of catechol estrogens. J. steroid Biochem. 18 (1983) 263-271. 14. Hospital M., Busetta B., Courseille C. and Precigoux G.: X-Ray conformation of some estrogens and their binding to uterine receptors. J. steroid Biochem. 6 (1975) 221-225.
15. Main P., Hull S. E., Lessinger L., Germain G., Declercq J. P. and Woolfson M. M. MULTAN 78-A system of computer programs for the automatic solution of crystal structures from X-ray diffraction data (Universities of York. England. and Louvain, Relaium). 16. Griffin J. F.,Duax W. L. and Weeks?. M. In Atlas of Steroid Structure. IFI/Plenum Press, New York, Washington, London (1984) pp. 7-13. 17. Hankins D., Moskowitz J. W. and Stallinger F. H.: Water molecule interactions. J. Chem. Phys. 53 (1971) 4-554. 18. Jeffrey G. A. and Lewis L.: Cooperative aspects of hydrogen bonding in carbohydrates. Carbohydrate Res. 60 (1978) 179-182.
J. steroid Bfodwm. Vol. 29, No. 4, pp. 387-392, 1988 Primal in Great Britain. AI1rights mcrved
STEROID STRUCTURE AND FUNCTION. MOLECULAR NONFORMATION OF 4-HYDRO~EST~DIOL AND ITS RELATION TO OTHER CATECHOL ESTROGENS ZDZISLAWWAwRzAKt, WILLIAM L. DUAX$, PHYLLISD. STRONOand JUDITH Wnxaz* Medical Foundation of Buffalo, Inc., 73 High Street, Buffalo, NY 14203and *Departments of Obstetrics and Gynecology and Pharmacology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, U.S.A. (Received 2 July 1987)
~~-Hydroxylation of estrogens at C(2) or C(4) effects ~fferent~By their binding afEnity to and dissociation rate from the estrogen receptor. The X-ray crystal structure of 4-hydroxyestradiol (COH-Er) is reported here and compared with that of 2-hydroxyestradiol (2-OH-Er), the 2- and ehydroxylated derivatives of estrone (E, ) and with that of the parent estrogens, El and Es. The overall molecular shape and hydrogen bonding patterns of each were examined for their possible relevance to their binding to the estrogen receptor and their biological activity. A shift in the B-ring conformation away from the symmetrical 7a,8/?-half-chair form toward the @-sofa form is induced by both 2- and Chydroxy substitution. This shift appears to be larger in the case of Es than Et derivatives and to be correlated with an observed change in the hydrogen bonding potential of the C(3) hydroxyl. In 4-OH-Er, as in Er and 4-OH-E,, the C(3) hydroxyl functions both as a hydrogen bond donor and acceptor. In contrast in 2-OH-E, the hydroxyl functions only as a donor. The markedly reduced afhnity of 2-hydroxylated estrogens for the estrogen receptor could be due to a combination of steric interactions, competition between O(2) and O(3) for hydrogen bonds for a common site on the receptor, and to general interference with hydrogen bond formation of O(3). The C(4) hydroxyl participates in the formation of a chain of hydrogen bonds in the solid state that is similar to a chain seen in single crystals of Er. The presence of a similar chain of hydrogen bonds involving O(3) in the receptor site could account for the decreased dissociation rate of the 4-OH-E, receptor complex.
~ODU~ION
Catecholestrogens are formed by hydroxylation of the estrogen molecule at either the C(2) or the C(4) position. In the body as a whole 2-hydroxyla~on predominates and represents a major route of metabolism of phenolic estrogens [ 11. Microsomal cytochrome P-45O-dependent monooxygenase(s) which catalyzes the reaction and acts primarily as a 2-hydroxylase has been identified in liver as well as in several steroidogenic and estrogen target organs [l-6]. However, hydroxylation at the C(4) position also occurs [2,7J and may be prominent in certain targets of estrogen action. Thus anterior pituitary and brain tissue have been reported to convert estradiol in uitro to its 2- and 4-hydroxylat~ metabolites in a 1: 1 ratio [I, 7’J. In addition, it has been recently demonstrated that catecholestrogen formation may be mediated by certain peroxidases and by microsomes, by an organic hydroperoxidedependent peroxidatic mechanism that also generates the 2- and 4-hydroxylated metabolites in a 1: 1 ratio [8,9]. tOn a leave from Technical University of Lodz, Lodz, Poland. $To whom correspondence should be addressed. 381
The physiolo~cal role of catecholestrogens formed in estrogen target tissues is not known. However, they have been proposed to act as autocrine or paracrine mediators of some of the ef&cts of the parent hormone. The addition of a second hydroxyl group to ring A of phenolic estrogens markedly alters their biochemical characteristics. By virtue of their catechol structure they can inhibit cateehol-Omethyltransferase and tyrosine hydroxylase and, thereby, potentially influence the rate of biosynthesis or degradation of catecholamines [I, lo]. Acting in this capacity the Z- and Chydroxylated estrogens appear equipotent [7]. Substitution of a 2- or ehydroxyl group also changes the interaction of the estrogen molecule with its receptor. However, in this respect it is possible to differentiate between the consequences of 2- and 4-hydroxylation. While both substitutions reduce the affinity of the steroid for the receptor this effect is greater for 2- than Chydroxylation [l 11. Moreover, 2- and Chydroxylation have opposite effects on the dissociation rate of the estrogen-receptor complex. Hydroxylation of C(2) substantially increases while that at C(4) significantly decreases the dissociation rate of the steroid. Consequently, 2-hydroxylated estrogens are weak estrogen agonists whereas the ~hyd~xylat~
ZDZISLAW WAWRZAK er al.
388
estrogens are potent and possibly long-acting estrogens. We have proposed that the steroid A-ring plays a critical role in initiating estrogen receptor binding and that the D-ring controls estrogen potencyjl21. The strength and orientation of hydrogen bonds involving O(3) is a dominant factor governing A-ring interaction. The X-ray crystallographic studies of 2and 4-hydroxy estrogens have been undertaken in order to determine the influence that these substitutions have upon the overall shape of the molecule and the strength and orientation of hydrogen bonds involving O(3). These studies were carried out in an effort to identify characteristic patterns in molecular conformation and hydrogen bonding interactions. The existence of such patterns would indicate an energetic preference or stability that would suggest the pattern of interactions found in the receptor site. We report here the results of the X-ray crystal structure analysis of 4-hydroxyestradiol (4-OH-Ez) together with a comprehensive analysis of the previously published structures of 2-OH-E2, 2-OH-E,, 4-OH-E, [13] and the parent compounds, E, and E, 1141.
EXPERIMENTAL
Crystals of 4-OH-E, were grown by slow evaporation from ethanol. Unit-cell parameters were determined by least-squares fit of the 8 values of 25 reflections. Intensity data were collected to f3 = 75” on an Enraf-Nonius CAD4 diffractometer using CuICcr radiation. The structure was solved by means of direct methods using the program MULTAN [ 151 and refined by full-matrix least-squares. All hydrogen atom positions were found from difference Fourier maps and refined with thermal parameters equal to those from corresponding nonhydrogen atoms. Nonhydrogen atoms were refined anisotropically. The crystal and refinement data are given in Table 1. The nonhydrogen atom coordinates and equivalent isotropic thermal parameters are collected in Table 2. Structure factors, atomic coordinates for the hydrogen atoms, anisotropic thermal parameters for nonhydrogen atoms and bond distances, bond angles and torsion angles for nonhydrogen atoms are available from the authors upon request.
Table
I. Crystal
and refinement
Molecular formula Molecular weight Crystal symmetry Space group Z Cell dimensions a, A
data
C,sH,,O, 288.4 Monoclinic p2, 2 7.0226(8) 19.2395(20) 5.7348(6) 107.806(2) 737.7(3)
Reliability factors Observed data All data
0.045/ I527 0.04611563
Table 2. Fractional atomic coordinates (x 104) and equivalent isotropic thermal parameters .&’ (x IO*) for nonhydrogen atoms with estimated standard deviations in parantheses Atom
x/a(c)
C(1) C(2)
462 l(4) 5108(4) 3662(4) 1748(4) 1246(3) - 836(4) - 1272(4) 548(3) 2238(4) 2729(4) 4074(4) 3550(4) 1944(3) 102(3) - 1541(4) - 1099(4) 951(4) 2760(5) 3916(3) 279(3) 2152(3)
C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(l0) C(l1) C(12) C(J3) C(14) C(15) C(16) C(17) C(l8) O(3) O(4) O(l7B)
y/b@ ) 76~2) 7104(2) 689 l(2) 7177(2) 7677(2) 7980(l) 8474(2) 8928 8455(2) 790 l(2) X867(2) 9453(2) 993 l(2) 9487(2) 10020(2) 10612(2) 10435(2) 10341(2) 6402(2) 6941(2) 11034(2)
z/c(a) 9661(5) 8 176(6) 6055(5) 5502(4) 6946(4) 6124(5) 7973(6) 9 190(4) 10721(4) 9101(4) 12231(6) 13740(5) 12 153(4) 10824(4) 97 1O(6) 11636(6) 13509(4) 10371(6) 4468(4) 3413(4) 14439f4)
B,(a) 328(7) 342(7) 302(6) 284(6) 266(6) 306(6) 353(7) 251(5) 277(6) 269(6) 385(7) 365(7) 249(5) 275(6) 388(8) 405(S) 285(6) 353(7) 400(6) 374(5) 357(5)
RESULTS
The overall geometry of the 4-OH-E2 molecule is shown in Fig. 1. When the geometry of 4-OH-E2 is compared with that of 2-OH-E2 [8], the bond lengths and angles are found to be in very good agreement. However, there is a small but significant difference in B-ring torsion angles and overall molecular shape. The distribution of conformations of the B-ring in estradiol, estrone, and their catechol derivatives [ 131, as a function of this ring symmetry is shown in Fig. 2. Generally, the B-ring conformation of 1,3,5(10)estratriene steroids vary from a 7ol,8/?-half-chair conformation in which atoms C(9), C( lo), C(5) and C(6) are coplanar and atoms C(7) and C(8) are equidistant from that plane on opposite sides (i.e. asymmetry
Fig. 1. A stereoscopic view of the 4-OH-E, molecule prepared using the program ORTEP. A stereo viewing device will permit the reader to see the molecule in three dimensions. Contact the authors for further information.
Catechol estrogen conformation and binding
70,BP
1 hoif
25 20 s
clwrir
i
p
15
‘V
h
u” Q
infmmtdiafe 5
10 i
I
0
5
10
d
15 20 25 AC2 (5-10)
30
Fig. 2. Relation of asymmetry parameters of the B-rings in 1,3,5~lO)~~at~ene structures. The rotational assets parameter AC@-IO) is plotted vs the mirror beets parameter AC*(S).E,(A), ZOH-E, (A), 4-OH-E, (V), E2 tO),2-OH-E, (0). 4-OH-& (W.
parameter 4<=,(5-lo)<7 [ill) to a 8/S-sofa conformation in which atoms C(9), C(lO), C(S), C(6) and C(7) are coplanar and C(8) is on the fi side of that plane (i.e. ~~rn~t~ parameter AC,(S)< 10 [16]). X-Ray diffraction studies of three crystal forms of estrone have been reported including one form containing two independent molecules in the crystallographic unit cell [14]. The conformation of estrone itself in three of the four observations (solid triangles in Fig. 2) is close to the symmetric 7a,8j?-half-chair form. The fourth conformer of estrone is si~ifi~ntly different and more nearly approximates the 8jS-sofa conformation. It is worth noting that the two conformers exhibiting the maximum difference are the pair that occur together in one crystal form. The 2- and Cbydroxy derivatives of estrone also crystallize with two independent molecules in the crystallographic unit cell. All derivatives of estrone have conformations lying midway betweeti these extremes with the 2-bydroxy derivatives being closest to the 8#?-sofa coronation. Tbe B-ring of estradiol determined in three independent observations (solid circles in Fig. 2) has a slightly distorted 7a,8J-half-chair conformation and *Average values for these angles in the nine structures in Fig. 2 having the 7a,8@-half-chair conformation (AC,(S)> 10.0 or AC+10)~ 10.0).
389
exhibits little variation. In contrast, the B-ring of 2-OH-E, (open circle) has the rarely observed perfect 8/I-sofa conformation. The B-ring in 4-OH-E2 (half circle) has a slightly distorted O/?-sofa conformation, i.e. shifted toward an intermediate 8/3-sofa-7a,8Bhalf-chair conformation. This shift in conformation toward the 8/l-sofa form with hydroxylation is apparently the result of the combined influence of the 17@and the 2- or Cbydroxy substitutions. The b-face interactions between the C(18) methyl group and the 17-hydroxy on one side and the axial bydrogens at C(8) and C(l1) on the other appear to cause the observed closing of the C(14)-C(8)-C(9)-C(ll) and C(S)-C(9)-C!(ll~(l2) torsion angles from 54 and 53’* to 51 and 50”, respectively. This twist about the C(8)-&(9) bond opens the C(7)-C(S)-C{9)-C( 10) torsion angle from 54” to 59” and shifts the B-ring toward the 8/3-sofa conformation. A preference for the 7a,8/?-half-chair conformation is illustrated in Fig. 3 for estradiol, estrone and the catechol derivatives of estrone. The observation of an 88-sofa conformation in only one of four crystallographically independent estrone structures suggests that, while this conformation is energetically feasible, it is less favored. Hydroxyl substitution in the 2- and 4-positions of estradiol appear to stabilize the 88-sofa conformation. The conformation of 4-OH-E, is intermediate between those of 2-OH-E, and estradiol (Fig. 3). Although the difference in conformation is subtle it may influence the interactive properties of the steroid surface or of the 3-bydroxyl group. Tbe angles around the C(3) of 4-bydroxyestradiol exhibit the characteristic distortion noted previously in 2-bydroxyestradiol, 4-bydroxyestrone and 2-hydroxyestrone[13]. In all four cases the largest angle is opposite the bond that is tram to the bydroxyl hydrogen [13]. In 4-OH-E, the two angles C(2j-C(3)-0(3) and C(4)-C(3)-0(3) differ by almost IO”. Tbe angles around the C(4) atom in 4-bydroxyestradiol do not follow this pattern. Tbe C(3)-C(4)O(4) and C(5~(4~(4) angle differ by less tban 1” and the distortion is in the opposite direction. The angle opposite the bond that is trans to the bydroxyl hydrogen is the smaller of the two. Tbe bydroxyl hydrogens are almost coplanar with the A-ring in 4-OH-E, where the torsion angles defining deviations from coplanarity are -2” for C(3)474)-0(4)-H(40) and 6” for C(2)-C(3)--0(3)-H(30). Values for
Fig. 3. Stereo view (see caption to Fig. I) of a least-squares fit of & (solid Ike) against 4-OH-E, and 2-OH-E, (dashed lines). The least-squares process minimized the separation between corresponding atoms in the C- and D-rings of the two structures.
ZDZISLAW WAWRZAK et al.
390
O(
Fig. 4. Hydrogen bond geometry of the A-ring of (a) 4-OH-E,, (b) &, (c) 4-OH-E, and (d) 2-OH-E,. Dashed lines (---) represent intermolecular hydrogen bonds and dot dashed lines (-.--) represent intramolecular hydrogen bonds. Acceptor and donor atoms on adjacent molecules include water and 0(17) oxygens. Note that the O(3) hydroxyl acts as a hydrogen bond donor and acceptor in (a), (b) and (c) but as a bifurcated donor only in (d).
2-OH-E2 are - 18” for C(lw(2)-0(2kH(20) and 5” for C(2)-C(3)-0(3)-H(30) [13]. The O(3) atom of 4-OH-E, accepts an intramolecular hydrogen bond from O(4) and donates a hydrogen bond to the 0( 17) of an adjacent molecule. The O(4) atom of 4-OH-E* accepts a hydrogen from the 0(17) of another nearby steroid (Fig. 4a). This hydrogen bonding pattern is compared with those observed in crystals of E,, 4-OH-E,, and 2-OH-E, in Fig. 4. DISCUSSION
X-Ray analysis reveals that 2- and 4-hydroxylation of estradiol and estrone induces subtle alterations in the overall shape of the steroid and much more obvious alterations of the hydrogen bonding capabilities of the C(3)-hydroxyl. The changes associated with the different substitutions offer insight into the molecular basis for the variation in binding and activity of the 2- and 4-hydroxy derivatives. Relatively poor binding of the 2-hydroxy derivatives of estrone and estradiol, despite almost identical conformations of 2-hydroxy- and 4-hydroxyestradiol,
suggest the O(2) hydroxyl may be directly responsible for the decreased binding affinity. There are two possible ways in which a 2-hydroxy substituent could interfere with receptor binding. Either the 2-hydroxy substituent would penetrate the receptor surface when the steroid is properly oriented or the 2-hydroxy would mimic the 3-hydroxy in its association with the receptor and generate a misorientation of the ligand. In the first case the 2-hydroxy would constitute a steric hindrance to binding and in the second case it might promote inappropriate and nonproductive binding. An association of the steroid with the receptor in which the 2-hydroxy substituent in 2-OH-E, mimicked the 3-hydroxy in E, (Fig. 5) would lack the proper orientation of the 17B-hydroxy substituent and contribute to the observed enhancement in the rate of dissociation. Both of these interpretations would be consistent with increased dissociation rates of the receptor complexes of 2-OH-E, and 2-OH-E*, compared to their parent steroid. In contrast to the effect of a 2-OH, the presence of a 4-OH substituent appears to be well tolerated by the receptor. Furthermore, the hydrogen bond pattern established between 4-OH-E, and the receptor
Catechol estrogen conformation
Fig. 5. View of a least-squares superposition of Es (solid line) and 2-OH-E, (dashed line) in which the Z-OH substituent mimics the 3-hydroxy group of estradiol. may strengthen the association and decrease the dissociation rate. Two interrelated aspects of the hydrogen bonding pattern observed in the solid state support this contention, the numbers of individualhydrogen bonds formed and their presumed strengths as reflected in their length and geometry. In 5 out of 7 estrone and estradiol structures the O(3) forms a strong hydrogen bond directing its hydrogen syn-periplanar to the C(2)-C(3) bond. The O(3) of estradiol always acts as an acceptor as well as a donor, whereas the O(3) of estrone acts as a donor only. Similarly, in 2-OH-E2 and 2-OH-E, the O(3) acts as a donor only and accepts no inter- or intramolecular hydrogen bonds. This could explain the lower dissociation rate of Ez relative to E, and 2-O&E,. In E, and E, the length of the hydrogen bonds in which O(3) acts as a donor ranges from 2.64 to 2.83A. In 2-OH-E, and 2-OH-E, the hydrogen bond lengths are significantly larger (2.93 and 3.08 A) and presumably weaker than the comparable bonds in estrone and estradiol. This observation is consistent with the lower affinity of the 2-hydroxy derivatives of E, and E, relative to their parent compounds. In contrast, in 4-OH-E, and both c~stallo~aphi~lly independent 4-0X-I-E, molecules the O(3) acts as an intramolecular hydrogen bond acceptor as well as an intermolecular hydrogen bond donor. Furthermore in 4-OH-E, [13], the 4-hydroxy acts as an inter-
and binding
391
molecular hydrogen bond acceptor as well. There is theoretical [ 17j and experimental evidence [ 181 that the formation of a chain of three hydrogen bonds such as this enhances the strength of these hydrogen bonds. The lengths (2.73, 2.74 and 2.75& and, presumably, strengths of the hydrogen bonds formed in the crystal structures of 4-OH-E, and 4-OH-E, are similar to those observed in estradiol and estrone. Increased stability due to the formation of more, shorter, and stronger hydrogen bonds and their compatibility with the apparent location of hydrogen bond acceptor and donor sites on the receptor may account for the higher binding affinity and the slower dissociation rate from the receptor of 4-OH-E, and 4-OH-E,. The data available on binding affinities and dissociation rates as well as the hydrogen bonding characteristics of estrone, estradiol and their 2and ~hydroxylated derivatives are surnrna~~ in Table 3. It should be noted that while the addition of a Chydroxy group to estrone permits the O(3) to act as an acceptor as well as a donor, the addition of a 2-hydroxy group to estradiol results in a loss of that ability. The behavior observed in the solid state is consistent with the effect of these substitutions on the binding affinity of these compounds. Acknowledgements-This work was supported in part by NIAAMDD Grant No. AM-26546 and_DDR Grant Nd.
RR-05716 fto WLD’I and bv NICHHD Grant HD-09743 (to JW). T&e authors would*like to thank G. Del Rel, M.
Tugac and J. Gallmeyer for their technical assistance. The organization and analysis of the data base associated with the investigation, and some of the illustrations were carried out using the PROPHET system, a unique national computer resource sponsored by the NIH.
REFERENCES
Ball P. and Knuppen R.: Catecholestrogens (2- and 4-hydroxy estrogens). Chemistry, biogenesis, metabolism, occurrences and physiological significance. Actu. endoer., Copenkt. 93 (1980) l-127. Hersey R. M., Williams K. I. and Weisx J.: Catechol estrogen formation by brain tissue: Characterization of a direct product isolation assay for estrogen 2- and 4-hydroxylase activity and its application to studies of 2. and 4-hydroxyestradiol formation by rabbit hypothalamus. Endocrinology 109 (1981) 1912-1920.
TabIc 3. Hydrogen bond geometry, binding affinity sod dissociation rate of estradiol (E.& cstrone (E, f and their 2-hvdroxv and 4-hvdroxv derivatives
-
Hydrogen bond geometry 0(3)D,. . A(&* 0(3)A.. . L&h)*
4.OH-E, E, 2-OH-E, 4-OH-E,
2.70(3) 2.86(2) 2.73 2.80(4) 2.82(3) 2.73
2.76(2) 2.65 2.80
Estrogen receptor binding Dissociation? fi (min) RBA ailinity 100 24 45 11 2 11
95.0 11.3 130.0 8.7 3.7 15.1
*Hydrogen bond distances represent average values with number of contributors in parentheses. D and A refer to donor and aeocptor atoms in the hydrogen bond. tData from ref. 1I, rate of dissociation at 25°C.
392
ZDZISLAWWAWRWK et al.
3. Hoffman A. R., Paul S. M. and Axelrod J.: Catecholestrogen synthesis and metabolism by human breast tumors in vitro. Cancer Res. 39 (1979) 45844587. 4. Mondschein J. !I., Hersey R. M., Dey S. K., Davis D. L. and Weisz J.: Catechol estrogen formation by the pig blastocyst during the preimplantation period. Biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity. Endocrinology 117 (1985) 2339-2346. 5. Hammond J. M., Hersey R. M., Walgoa M. A. and Weisz J.: Catecholestrogen production by porcine ovarian cells. Endocrinology 118 (1986) 2292-2299. 6. Fishman J. and Norton B.:Catecholestrogen formation in the central nervous system of the rat. &docrinology 69 (1985) 1054-1059. I. Ball P., Haupt M. and Knuppen R.: Biogenesis and metabolism of catecholestrogens in vitro. In Catechol Estrogens (Edited by G. R. Merriam and M. Lipsett). Raven Press, New York (1983) 91-104. 8. Mondschein J. S., Hersey R. M. and Weisz J.: Purification and characterization of estrogen-2/ 4-hvdroxvlase activitv from rabbit hvnothalmi: per&da&z-mediated -&echo1 estrogen formation. EndocrinoloPv 119 (1986) 1105-l 112. 9. Weisz J. a;d Bui‘ Q.: ‘Multiple pathways of catecholestrogen formation: Characterization of NADPHand organic hydroperoxide-dependent 2- and 4-hydroxylation of estradiol by microsomes from human placenta and rat brain. 69rh Annual Meeting of the Endocrine Society, Indianapolis, Indiana, June 1987. Abstr. No. 782. 10. Lloyd T. and Weisz J.: Direct inhibition of tyrosine hydroxylase activity by catecholestrogen. J. biol. Chem. 253 (1978) 4841-4843.
11. MacLusky N. J., Bamea E. R., Clark C. R. and Naftolin E.: Catechol estrogens and estrogen receptors. In Catechol Estrogens (Edited by G. R. Merriam and M. B. Lipsett). Raven Press, New York (1983) pp. 151-165. 12. Duax W. L. and Weeks C. M.: Molecular basis of estrogenicity: X-Ray crystallographic studies. In Estrogens in the Environment (Edited by J. W. McLachlan). Elsevier North-Holland, New York (1980) pp. 11-31. 13. Daux W. L., Griffin J. F., Swenson D. C., Strong P. D. and Weisz J.: Steroid structure and function-IX. Molecular conformation of catechol estrogens. J. steroid Biochem. 18 (1983) 263-271. 14. Hospital M., Busetta B., Courseille C. and Precigoux G.: X-Ray conformation of some estrogens and their binding to uterine receptors. J. steroid Biochem. 6 (1975) 221-225.
15. Main P., Hull S. E., Lessinger L., Germain G., Declercq J. P. and Woolfson M. M. MULTAN 78-A system of computer programs for the automatic solution of crystal structures from X-ray diffraction data (Universities of York. England. and Louvain, Relaium). 16. Griffin J. F.,Duax W. L. and Weeks?. M. In Atlas of Steroid Structure. IFI/Plenum Press, New York, Washington, London (1984) pp. 7-13. 17. Hankins D., Moskowitz J. W. and Stallinger F. H.: Water molecule interactions. J. Chem. Phys. 53 (1971) 4-554. 18. Jeffrey G. A. and Lewis L.: Cooperative aspects of hydrogen bonding in carbohydrates. Carbohydrate Res. 60 (1978) 179-182.