Dehydroepiandrosterone and Dihydrotestosterone Recognition by Human Estrogenic 17β-Hydroxysteroid Dehydrogenase

Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein. Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. TheK m of estrone is more than 1000-fold lower than that of DHEA and the K m of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17β-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 → Val variant showed a significantly decreased K m for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a β-oriented form, which approaches the transition-state conformation for DHEA reduction. Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein. Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. TheK m of estrone is more than 1000-fold lower than that of DHEA and the K m of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17β-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 → Val variant showed a significantly decreased K m for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a β-oriented form, which approaches the transition-state conformation for DHEA reduction. dihydrotestosterone dehydroepiandrosterone estrone estradiol 17β-hydroxysteroid dehydrogenase Steroid hormones are involved in a wide variety of physiological functions, including metabolism, reproduction, aging, and cancer. Steroid hormones follow a common pathway; they diffuse into cells and bind to their receptors, which in turn bind hormone response elements to the DNA and interact with the transcriptional machinery to activate or repress transcription of target genes (1.Milgrom E. Baulieu E.E. Kelly P.A. Hormones From Molecules to Disease. Hermann, New York1990: 405-420Google Scholar, 2.Fawell S.E. Lees J.A. White R. Parker M.G. Cell. 1990; 60: 953-962Abstract Full Text PDF PubMed Scopus (486) Google Scholar). One important mechanism by which cells can modify hormone action is the modulation of the steroid structure itself (3.Andersson S. J. Endocrinol. 1995; 146: 197-200Crossref PubMed Scopus (53) Google Scholar). Steroid hormones share a very similar structure that has as its core the cyclo-pentenophenanthrene ring. The addition of an 18th carbon atom results in the estrogen ring (C-18), the addition of a 19th carbon atom results in the C-19 ring (including androgens such as DHT1 and testosterone), and the addition of a two-carbon lateral chain produces the pregnane ring (C-21). Despite their very similar structures, steroid hormones have significantly different physiological activities. DHT, the most potent androgen, estradiol (E2), the most potent estrogen, and DHEA, the most abundant hormone precursor in humans and other mammals, exert numerous and diverse actions on their target tissues (Fig. 1) (4.Thomas D.B. Cancer. 1984; 53: 595-601Crossref PubMed Scopus (164) Google Scholar, 5.Shealy C.N. Integr. Physiol. Behav. Sci. 1995; 30: 308-313Crossref PubMed Scopus (55) Google Scholar, 6.Griffiths K. Morton M.S. Nicholson R.I. Eur. Urol. 1997; 32: 24-40PubMed Google Scholar). The detailed mechanism of steroid discrimination has not been well elucidated up to now. The enzymes of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family are responsible for the last step in the formation of all androgens and estrogens. The reduction of the 17-ketone to a 17β-hydroxyl increases the affinity of the steroids to their cognate receptors (7.Peltoketo H. Lsomaa V. Poutanen M. Vihko R. J. Endocrinol. 1996; 150 suppl.: S21-S30PubMed Google Scholar). The 17β-reduction activity of these enzymes is thus required for the synthesis of all active androgens and estrogens. Molecular cloning has revealed the presence of seven isozymes of 17β-HSD, six of which are members of the short chain dehydrogenase/reductase superfamily (8.Persson B. Krook M. Jornvall H. Eur. J. Biochem. 1991; 200: 537-543Crossref PubMed Scopus (411) Google Scholar, 9.Nokelainen P. Peltoketo H. Vihko R. Vihko P. Mol. Endocrinol. 1998; 12: 1048-1059Crossref PubMed Google Scholar). 17β-HSD1 primarily catalyzes the interconversion of estrone (E1) and estradiol, but it also has some catalytic activity for the interconversion between DHEA and 5-androstene-3,17-diol, 4-androgene-3,17-dione and testosterone, as well as between A-dione and DHT (9.Nokelainen P. Peltoketo H. Vihko R. Vihko P. Mol. Endocrinol. 1998; 12: 1048-1059Crossref PubMed Google Scholar, 10.Labrie F. Simard J. Luu-The V. Pelletier G. Belghoni K. Bélanger A. Sheppard M.C. Stewart P.M. Hormones, Enzymes and Receptors. 8. Baillieres's Tindall Ltd., London1994: 451-474Google Scholar), thus constituting a good model to study C-18/C-19 steroid or estrogen/androgen discrimination. We have previously proved that 17β-HSD1 is a dimer consisting of two identical subunits (11.Lin S.-X. Yang F. Jin J.Z. Breton R. Zhu D.W. Luu-The V. Labrie F. J. Biol. Chem. 1992; 267: 16182-16187Abstract Full Text PDF PubMed Google Scholar). Immunochemical analyses have confirmed the presence of this enzyme in human placenta, breast, and ovary granulosa cells (12.Luu-The V. Labrie C. Simard J. Lachance Y. Zhao H.F. Conët J. Leblanc G. Labrie F. Mol. Endocrinol. 1990; 4: 268-275Crossref PubMed Scopus (182) Google Scholar, 13.Sawetawan C. Milewich L. Word R.A. Carr B.R. Rainey W.E. Mol. Cell. Endocrinol. 1994; 99: 161-168Crossref PubMed Scopus (86) Google Scholar). Since it is well known that estradiol stimulates the proliferation of mammary tumor cells, this enzyme is an important target for breast cancer therapy (14.Lippman M.E. Breast Cancer Res. Treat. 1986; 7: 59-70Crossref PubMed Scopus (231) Google Scholar, 15.Labrie F. Luu-The V. Lin S.-X. Labrie C. Simard J. Breton R. Belanger A. Steroids. 1997; 62 (0): 148-158Crossref PubMed Scopus (421) Google Scholar). The structure of 17β-HSD1 from human placenta was first determined at 2.2 Å resolution (16.Ghosh D. Pletnev V.Z. Zhu D.W. Wawrzak Z. Duax W.L. Pangborn W. Labrie F. Lin S.-X. Structure. 1995; 3: 503-513Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). More recently, the structure of the complex of 17β-HSD1 with E2, as well as the enzyme structure in the presence of E2 and NADP, have been reported (17.Azzi A. Rehse P. Zhu D.W. Campbell R. Labrie F. Lin S.-X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar, 18.Breton R. Housset D. Mazza C. Fontecilla-Camps J. Structure. 1996; 4: 905-915Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Nevertheless, it is still unclear why a small structural modification, like the addition of a C-19 methyl group, can result in a very different catalytic activity, and how steroids are recognized by different isozymes and receptors. To answer these questions, we have determined and compared the structures of 17β-HSD1 in complex with different steroids. Here we report the crystal structures of 17β-HSD1 complexed with either DHEA or DHT. The comparison of the structures of DHEA, DHT, and E2 complexes with 17β-HSD1 gives an informative picture of important steroid hormone recognition and discrimination by the enzyme. DHEA, DHT, 17β-estradiol, and estrone were purchased from Aldrich. NAD, NADH, β-octylglucoside, dithiothreitol, polyethylene glycol 4000, and glycerol were from Sigma. We packed the Q-Sepharose ion exchange and the Blue-Sepharose CL-6B columns ourselves with media and columns from Amersham Pharmacia Biotech (Montreal, Canada). Phenyl-Superose HR 10/10 was purchased directly from the same company. All reagents were of the best grade available.Spodoptera frugiperda cells (Sf9 cells), Bac-N-BlueTM transfection kit, and transfer vector pBlueBac4.5 were purchased from Invitrogen Corp.; Grace's insect cell culture medium, yeastolate, and lactalbumin hydrolysate were from Life Technologies, Inc.; cell culture grade fetal bovine serum was from HyClone. 17β-HSD1 was purified from human placenta with a previously described rapid purification procedure (11.Lin S.-X. Yang F. Jin J.Z. Breton R. Zhu D.W. Luu-The V. Labrie F. J. Biol. Chem. 1992; 267: 16182-16187Abstract Full Text PDF PubMed Google Scholar). The crystallization was carried out with the vapor-diffusion technique in hanging drops. Crystals were obtained in several days with 30% polyethylene glycol 4000, 0.06% (w/v) β-octylglucoside, 0.12 m MgCl2, 0.1m Hepes, pH 7.5, and in the presence of saturating DHEA or DHT (1 mm). In the absence of glycerol, 17β-HSD1 gets inactivated readily at low temperature (at less than 10–15 °C), and it gets reactivated by warming at 30 °C where the enzyme is stable (29.Jarabak J. Seeds A.E. Talalay P. Biochemistry. 1996; 5: 1269-1278Crossref Scopus (105) Google Scholar). The enzyme stability is necessary for obtaining high quality crystals with a high occupancy of substrate. We thus attempted the crystallization at 27 °C, which yielded good results. 2Q. Han and S.-X. Lin, unpublished results. The data for the DHEA complex was collected on an R-axis IIC image plate area detector on a Rigaku RU-H2R rotating anode generator equipped with a graphite monochromator and a 0.5-mm collimator at room temperature. Data for the DHT complex were collected at −150 °C at beamline X12-C at Brookhaven National Laboratory. The two data sets were processed using DENZO and Z. W. in of Data in New Scholar, data sets were with the and E2 complex structures and were with New using the of the structure of the E2 complex as a was using the M. A. 1991; PubMed Scopus Google Scholar). The was with data between 10 and Å resolution and was to the high resolution of data structures were with good The electron for DHEA and DHT are well and they have been and with The electron for the residues and for the between residues and are than in the first enzyme structure and in the estradiol complex (17.Azzi A. Rehse P. Zhu D.W. Campbell R. Labrie F. Lin S.-X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar), but they are still not to the of residues of the by of the model to the high for the structure of the DHT and V. 5: Google from In the core In the All in a data for the 17β-HSD1 with DHEA and DHT and for the 17β-HSD1 with E2, DHEA, and DHT were obtained by of reduction at °C by the at The pH 7.5, and steroid for for estradiol and for The cell obtained from the and from the were and at for to the and in for the enzyme activity for steroids. were to the K steroids was obtained the same and were determined by the of Biochem. PubMed Scopus Google Scholar). was using a procedure with S. S. Biochem. PubMed Scopus Google Scholar). The activity was as described previously (11.Lin S.-X. Yang F. Jin J.Z. Breton R. Zhu D.W. Luu-The V. Labrie F. J. Biol. Chem. 1992; 267: 16182-16187Abstract Full Text PDF PubMed Google Scholar). The human 17β-HSD1 was from the vector R. Yang F. Lin B. Labrie F. Lin S.-X. J. Steroid Biochem. Mol. Biol. 1994; PubMed Scopus Google by chain to the vector for site-directed The to the variant was by using the site-directed mutagenesis the The two and the by the transfer vector was in cells with DNA the described by the Bac-N-BlueTM F. Invitrogen Scholar). cells were as at 27 °C in Grace's insect cell culture fetal serum and 10 were with at a of of for and at a of of for were with in (1 and at °C for In these the core of the the same structure as that of the enzyme (16.Ghosh D. Pletnev V.Z. Zhu D.W. Wawrzak Z. Duax W.L. Pangborn W. Labrie F. Lin S.-X. Structure. 1995; 3: 503-513Abstract Full Text Full Text PDF PubMed Scopus (257) Google and as that of the complex (17.Azzi A. Rehse P. Zhu D.W. Campbell R. Labrie F. Lin S.-X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar). The quality of the electron density for DHEA and DHT the of their DHEA and DHT, like E2, are in the hydrophobic that is at the of and cavity is composed of and and and and and The binding is Å by Å in and Å of the of the also the that is in all members of the short chain dehydrogenase/reductase superfamily and for the The atom of DHEA that of two with the protein. and in the active and a similar to that between the steroid and the catalytic residues in the The atom in DHEA two with of and of The between and is as by the of the chain of which has a high and a electron The with is and important for the bound substrate. The is also in the and a similar is in the complex of dehydrogenase with H. M. Structure. 1997; 5: Full Text Full Text PDF PubMed Scopus Google Scholar). the structures of the complexes of 17β-HSD1 are it is that the core of DHEA by about an the of the steroid binding and Å at the and Å at the as compared with the of E2 in the E2 complex DHT Å at the and Å at the but the core of DHT only results are it was that the binding the steroid (16.Ghosh D. Pletnev V.Z. Zhu D.W. Wawrzak Z. Duax W.L. Pangborn W. Labrie F. Lin S.-X. Structure. 1995; 3: 503-513Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). In all structures of DHEA, DHT, and E2, the is in a with the chain of In the E2 structure, the between E2 and the of Leu-149 is is a C-19 to of DHEA and DHT, these steroids at the same to The of Leu-149 is by two chain with and The electron density of Leu-149 is well and the of its and are the for the protein. The of Leu-149 with DHEA and DHT is to and of Leu-149 by Å from their in the presence of of of the active the in of DHEA and DHT. with E2 the different binding of DHT and DHEA result in of these steroids with several important residues in the binding they can make with the more and The E2 complex structure and study and S.-X. Lin, unpublished results. have revealed that an important in E2 chain with the of the with DHEA and DHT are we that the chain of in the DHEA and DHT complexes has a from its binding in the E2 complex and that its has to the hydrophobic of between 17β-HSD1 and the of the steroid to the binding of to the affinity of The for the between the binding of DHEA and of DHT by the in their The and of DHT are in the same while the between and in DHEA the and toward the DHT with and than DHEA, while DHEA with and a with of The of most of the active residues are but the (16.Ghosh D. Pletnev V.Z. Zhu D.W. Wawrzak Z. Duax W.L. Pangborn W. Labrie F. Lin S.-X. Structure. 1995; 3: 503-513Abstract Full Text Full Text PDF PubMed Scopus (257) Google has that different for and in the presence of (Fig. The of which several residues in the binding important for steroid of the of DHEA, DHT and E2 from The or 17-ketone in steroids are to other than the or The bound of DHEA and DHT are more similar to other than they are to the bound of image for the binding of can obtained from the steroid electron density in the different complex The of E2 in the binding was by its high of electron In the electron of DHEA and DHT are than that of E2 in the cognate they were in the presence of of DHEA and DHT than the that was in the of E2 (17.Azzi A. Rehse P. Zhu D.W. Campbell R. Labrie F. Lin S.-X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar). The of the DHEA and DHT complexes also that their are less well than was E2 in the binding of these steroids with 17β-HSD1 were carried out in to with the structural from that m for DHEA is more than 1000-fold than that for and the K m for DHT is about than that for estradiol, in with the structural of 17β-HSD1 with different K m and for a of two m and for estrogens with 17β-HSD1 were from in a The K m and for a of two m and for estrogens with 17β-HSD1 were from The catalytic mechanism of 17β-HSD1 for estrone reduction is to the transfer of a ion from the of the to the of estrone to a The in an with the in a toward the binding of 17β-HSD1 (17.Azzi A. Rehse P. Zhu D.W. Campbell R. Labrie F. Lin S.-X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar, C. Breton R. Housset D. Fontecilla-Camps J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of DHT and DHEA the between of the steroid and of the study using the model from (17.Azzi A. Rehse P. Zhu D.W. Campbell R. Labrie F. Lin S.-X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google and the from Breton R. Housset D. Mazza C. Fontecilla-Camps J. Structure. 1996; 4: 905-915Abstract Full Text Full Text PDF PubMed Scopus (191) Google that the is Å for E2, Å for DHT, and Å for these are 17β-HSD1 the and these we a lower of transfer for the C-19 steroids. we that the affinity of DHEA and DHT for 17β-HSD1 is with their K m and that the between and of is in with their compared with a result of the K m modification, the between the estrogens and C-19 steroids between and The discrimination between these steroids is thus all the steroid only the estrogens and E2 the C-19 methyl 17β-HSD1 is well for its high toward estrogen Although the binding cavity is almost entirely composed of hydrophobic residues nonspecific interactions, the arrangement of residues highly only for the to the small changes in the shape of the hormones can significantly affect the binding and affinity in critical hormone recognition and The between C-19 and the hydrophobic Leu-149 to the most important for C-19 steroids and in from binding in an the shape of the binding a critical role in steroid hormone discrimination. In with the site-directed mutagenesis was carried out to the variant by the Leu-149 by the very similar but from of the structure, the catalytic and discrimination were significantly in the the similar for E2 and TheK m of variant enzyme for DHEA has decreased from to while m for has from to the variant enzyme showed a lower K m for DHT as compared with the the is not as in the of DHEA changes to to some of with estrogens and to the of with C-19 steroids and thus are in good with the structural In we can that the of the Leu-149 → Val variant for estrogens while these significantly for C-19 in a of C-18/C-19 steroid discrimination. In the for C-19 steroids and estrogens similar results the role of the Leu-149 in the recognition of 17β-HSD1. The structure of 17β-HSD1 complexed with DHEA gives important about how the enzyme catalyzes the of the 17-ketone to the The electron density that the is toward the The was with an DHEA model with in which conformation and are toward the of the conformation is by the presence of the methyl the and not into the electron density and was from a in which it with and structures of DHEA a of of the compared with the of the steroid core J. Chem. 1995; Scopus Google Scholar). the conformation of was to the density and were and toward the The was by compared with its in the and small In this the 17-ketone can with and that the of and can the of the 17-ketone of DHEA, to its and to a this high thus the transfer from to and the transfer from ring to of the The presence of the chain of in to the also the of the β-oriented and in the structure of the E2 the of estradiol are with and the β-oriented the of the of and this is for of the substrate. The of a β-oriented is an step in the formation of the it the for the of 17-ketone to the present results a catalytic mechanism that a the reduction of DHEA, and this also to the reduction of other hormones (Fig. comparison of The model of DHEA is the structure, which is very similar to the small crystal structure of DHEA J. Chem. 1995; Scopus Google Scholar). The conformation of the model of 5-androstene-3,17-diol, the of the reduction of DHEA was using by to the conformation of The conformation of and of DHEA changes from toward an β-oriented the reduction The model of DHEA is also image The present structure and of have determined that a 17β-hydroxyl or 17-ketone in the is for binding with 17β-HSD1 Cancer. 1996; 3: Scopus Google Scholar). is with the formation of with and as in the the of the core of DHEA and DHT have been in the binding the to have are highly and are important in the of the binding while the are important for the binding affinity of steroids. The 17β-HSD1 complex structures the general of steroid hormone recognition by the The steroid binding to have The first the steroid the and residues that with The to the hydrophobic core of the to the the binding of The catalytic the and the catalytic that is on the of the steroid and a with The of the steroid is to the to estrogens and androgens have and or the critical residues that are to for the estrogenic and members of the family the hydrophobic binding In some residues the of the in an binding or binding at In such as a hydrophobic cavity that can interact with the We that the hydrophobic of more than that of as it similar activity with and C-19 We that other C-19 steroids and have similar with 17β-HSD1 as DHEA and DHT. is in with which that these C-19 steroids have similar K m and K for The of and hydrophobic in the of the steroid binding in 17β-HSD1 is to a common in other are for the significantly to binding were in the between the estrogen and Z. L. J.A. M. 1997; PubMed Scopus Google Scholar). of all cancer are sex including breast, and enzymes are thus for approaches on of the isozymes have than the this family of enzymes is an target for the of potent and to W.L. D. Struct. Biol. 1996; PubMed Scopus Google Cancer. 1996; 3: Scopus Google Scholar). The 17β-HSD1 complex structures suggest several to potent The hydrophobic binding the for the best to the a hydrophobic ring structure like that of E2 as the core of an β-oriented electron present to with and In an hydrophobic at or to the from Despite the of the hydrophobic to to at the of the with have been and to 17β-HSD1 C. C. C. Y. Labrie F. Cancer Res. 1992; Google Scholar). The of an toward or estrogenic by the shape of its hydrophobic in the C-19 of the its affinity toward 17β-HSD1 while the affinity toward We F. Labrie for in this We A. for with the and data We are also to the at Brookhaven National for with data We S. for the We also for in the

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.