Structure of the Ternary Complex of Human 17Я-Hydroxysteroid
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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 840–845, February 1999 Biochemistry Structure of the ternary complex of human 17b-hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin) and NADP1 MARK W. SAWICKI*, MARY ERMAN†,TERHI PURANEN‡,PIRKKO VIHKO‡§, AND DEBASHIS GHOSH*†¶ *Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263; †Hauptman-Woodward Medical Research Institute, 73 High Street, Buffalo, NY 14203; ‡Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health, University of Oulu, FIN-90220, Finland; and §Department of Biosciences, Division of Biochemistry, P.O. Box 56, FIN-00014 University of Helsinki, Finland Communicated by Herbert Hauptman, Hauptman-Woodward Medical Research Institute, Buffalo, NY, December 4, 1998 (received for review October 14, 1998) b ABSTRACT Excess 17 -estradiol (E2), the most potent of has been proposed to be involved in maintaining high E2 levels human estrogens, is known to act as a stimulus for the growth found in breast tumors of postmenopausal women (ref. 17, and of breast tumors. Human estrogenic 17b-hydroxysteroid de- references therein). A direct correlation between higher con- hydrogenase type 1 (17b-HSD1), which catalyzes the reduc- centrations of E2 and onset of breast cancer, especially in tion of inactive estrone (E1) to the active 17b-estradiol in postmenopausal women, is well established (ref. 17, and y breast tissues, is a key enzyme responsible for elevated levels references therein). There are reports of elevated E2 E1- of E2 in breast tumor tissues. We present here the structure of concentration ratios in breast tumors in comparison with the 1 y the ternary complex of 17b-HSD1 with the cofactor NADP E2 E1 ratio in circulating blood (18). Furthermore, a number and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an of studies appear to indicate higher levels of 17b-HSD1 equine estrogen used in estrogen replacement therapy. The activities in the outer quadrant of the breast, where tumors ternary complex has been crystallized with a homodimer, the most commonly occur (18–20). 17b-HSD1, therefore, poses an active form of the enzyme, in the asymmetric unit. Structural attractive target for structure-based rational drug design for and kinetic data presented here show that the 17b-HSD1- the prevention and control of breast tumor growth. catalyzed reduction of E to E in vitro is specifically inhibited Detailed knowledge of the biochemistry and molecular 1 2 b by equilin. The crystal structure determined at 3.0-Å resolu- biology of 17 -HSD1 has grown rapidly in the last 5 years, tion reveals that the equilin molecule is bound at the active site culminating in the determination of the three-dimensional in a mode similar to the binding of substrate. The orientation structure of the human enzyme (21). This has led to an of the 17-keto group with respect to the nicotinamide ring of atomic-level description of the E2-binding pocket of the en- NADP1 and catalytic residues Tyr-155 and Ser-142 is different zyme and understanding of its mechanism of action and the b molecular basis for the estrogen specificity of the enzyme from that of E2 in the 17 -HSD1–E2 complex. The ligand and b y (8–10). In addition, complexes of 17 -HSD1 with E2 and or substrate-entry loop densities are well defined in one subunit. 1 The substrate-entry loop adopts a closed conformation in this NADP (22, 23), various mutant complexes (24), and struc- subunit. The result demonstrates that binding of equilin at the ture-function analysis through site-directed substitutions and enzyme chimeras (9, 10) have been published that further active site of 17b-HSD1 is the basis for inhibition of E1-to-E2 clarify the mechanism of action and provide additional de- reduction by this equine estrogen in vitro. One possible y outcome of estrogen replacement therapy in vivo could be tailed insights into the origin of estrogenic androgenic spec- ificities of the enzyme. 17b-HSD1 belongs to the short-chain reduction of E2 levels in breast tissues and hence the reduced y risk of estrogen-dependent breast cancer. dehydrogenases reductase family (25), requiring a Tyr-X-X- X-Lys motif and a Tyr-Lys-Ser catalytic triad for activity (21, b b 26). 17 -Hydroxysteroid dehydrogenases (17 -HSDs) are a group We present here the structure of an inhibited ternary of enzymes that are involved in interconversion of active and complex of 17b-HSD1 with NADP1 and 3-hydroxyestra- inactive forms of androgens and estrogens (1–7) by 1,3,5,7-tetraen-17-one (equilin), which shows that the equilin NAD(P)(H)-linked oxidoreductive transfer of a hydride to and b b molecule binds at the catalytic site of 17 -HSD1. Equilin is one from the 17-position of steroid molecules. Six distinct 17 - of the major components of estrogens used in estrogen re- HSD isozymes, numbered 1–6, have been identified and cloned placement therapy, along with estrone and 17a-dihydroequilin. (2–7). These isozymes differ in specificities for substrate and These conjugated estrogens are administered under the com- tissue and in the preferred direction of the reaction. In human mercial name Premarin as salts of their sulfate esters, which are b breast tissues, the most active estrogen, 17 -estradiol (E2), is subsequently hydrolyzed to free estrogens. Kinetic data pre- formed by reduction of the inactive estrogen, estrone (E1), sented also supports in vitro inhibition of the enzyme’s E -to-E b b 1 2 which is catalyzed by 17 -HSD type 1 (17 -HSD1). The reduction activity by equilin. estrogenic specificity of 17b-HSD1 as well as its preference for the reduction reaction has been well established (8–10). 17b-HSD1 is expressed in steroidogenic tissues including MATERIALS AND METHODS estrogen target tissues such as normal and malignant endo- For crystallization experiments, human 17b-HSD1 was ex- metrium and breast tissues (11–16). Because of its estrogenic pressed in Sf9 insect cells as described (9, 10). The expressed specificity and preference for the E1-to-E2 reduction reaction, the enzyme is considered to be primarily responsible for E2 Abbreviations:HSD, hydroxysteroid dehydrogenase; 17b-HSD1, 17-b biosynthesis in gonads and in peripheral tissues. This enzyme HSD type 1; E1, estrone; E2,17b-estradiol, equilin, 3-hydroxyestra- 1,3,5,7-tetraen-17-one. The publication costs of this article were defrayed in part by page charge Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, Biology Department, Brookhaven National Lab- payment. This article must therefore be hereby marked ‘‘advertisement’’ in oratory, Upton, NY 11973 (PDB ID code 1EQU). accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. e-mail: ghosh@hwi. PNAS is available online at www.pnas.org. buffalo.edu. 840 Downloaded by guest on September 26, 2021 Biochemistry: Sawicki et al. Proc. Natl. Acad. Sci. USA 96 (1999) 841 enzyme was purified by using red-agarose affinity chromatog- Table 1. Kinetic data for inhibition of 17b-HSD1 by Equilin 1 raphy in the presence of 0.25 mM NADP followed by Mono Concentration, Inhibition, Q (Pharmacia) anion-exchange chromatography. The equilin Steroid Values mM % 6 SD P inhibition assays were performed by transiently expressing the 6 full-length human 17b-HSD1 cDNA in human embryonic Equilin 1 77.2 2.1 0.0001 6 kidney 293 cells under the cytomegalovirus (CMV) promoter 10 97.6 0.4 0.0001 by using a pCMV6 vector (27). The reductive activity of 17b-HSD1 was measured in cultured cells with 0.2 mM 3H- This holo-form represents a true ternary complex of the labeled substrate, using a 2-hr incubation time. To analyze the wild-type enzyme, with the cofactor and a steroidal ligand. inhibitory effects of equilin (Steraloids, Wilton, NH) on 17b- The kinetic data illustrating the ability of equilin to inhibit b HSD1 activity, the compound was added into the reaction the E1-to-E2 conversion by 17 -HSD1 is shown in Table 1. A b mixture at final concentrations of 1 mM and 10 mM. Amounts 77% inhibition of normal 17 -HSD1 activity (E1-to-E2) was of the substrate converted were determined from triplicate achieved with 1 mM equilin, establishing it as a potent inhibitor measurements of at least three independent experiments. of E2 synthesis. 1 Student’s t-test was used to analyze the significance of differ- Ligand-Binding Interactions. Both equilin and NADP ences between the activity results obtained with or without have well-defined electron density in the A subunit of the competing equilin. The kinetic data are summarized in Table 1. dimeric enzyme. However, the ligand density in the B subunit The enzyme was crystallized by vapor diffusion from 28% is poorly defined; therefore, the ligand was not included in the polyethylene glycol 4000 in Hepes buffer (pH 7.5) containing B subunit. The active-site structure of the 17b-HSD1-equilin 5 1 1 mM equilin. The space group was P212121 with a 44.02 Å, complex for the A subunit is shown in Fig. 1. The NADP and b 5 114.16 Å, and c 5 114.84 Å, with a complete dimer in the equilin molecules as well as amino acids responsible for asymmetric unit. The data was collected at an R-AxisIIc binding the ligand are shown in their electron densities. The image-plate detector under cryogenic conditions. The crystal density for equilin is shown before its inclusion in the protein ('0.1 mm 3 0.1 mm 3 0.15 mm) was flash-frozen in vapors model.