Structure of the ternary complex of human 17β-hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin) and NADP ⁺

Abstract

Excess 17␤-estradiol (E 2 ), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17␤-hydroxysteroid dehydrogenase type 1 (17␤-HSD1), which catalyzes the reduction of inactive estrone (E 1 ) to the active 17␤-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E 2 in breast tumor tissues. We present here the structure of the ternary complex of 17␤-HSD1 with the cofactor NADP ؉ and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an equine estrogen used in estrogen replacement therapy. The ternary complex has been crystallized with a homodimer, the active form of the enzyme, in the asymmetric unit. Structural and kinetic data presented here show that the 17␤-HSD1-catalyzed reduction of E 1 to E 2 in vitro is specifically inhibited by equilin. The crystal structure determined at 3.0-Å resolution reveals that the equilin molecule is bound at the active site in a mode similar to the binding of substrate. The orientation of the 17-keto group with respect to the nicotinamide ring of NADP ؉ and catalytic residues Tyr-155 and Ser-142 is different from that of E 2 in the 17␤-HSD1-E 2 complex. The ligand and substrate-entry loop densities are well defined in one subunit. The substrate-entry loop adopts a closed conformation in this subunit. The result demonstrates that binding of equilin at the active site of 17␤-HSD1 is the basis for inhibition of E 1 -to-E 2 reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E 2 levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.17␤-Hydroxysteroid dehydrogenases (17␤-HSDs) are a group of enzymes that are involved in interconversion of active and inactive forms of androgens and estrogens (1-7) by NAD(P)(H)-linked oxidoreductive transfer of a hydride to and from the 17-position of steroid molecules. Six distinct 17␤-HSD isozymes, numbered 1-6, have been identified and cloned (2-7). These isozymes differ in specificities for substrate and tissue and in the preferred direction of the reaction. In human breast tissues, the most active estrogen, 17␤-estradiol (E 2 ), is formed by reduction of the inactive estrogen, estrone (E 1 ), which is catalyzed by 17␤-HSD type 1 (17␤-HSD1). The estrogenic specificity of 17␤-HSD1 as well as its preference for the reduction reaction has been well established (8-10).17␤-HSD1 is expressed in steroidogenic tissues including estrogen target tissues such as normal and malignant endometrium and breast tissues (11-16). Because of its estrogenic specificity and preference for the E 1 -to-E 2 reduction reaction, the enzyme is considered to be primarily responsible for E 2 biosynthesis in gonads and in peripheral tissues. This enzyme has been proposed to be involved in maintaining high E 2 levels found in breast tumors of postmenopausal women (ref. 17, and references therein). A direct correlation between higher concentrations of E 2 and onset of breast canc...

Structure of Equilenin at 100 K: an estrone-related steroid

“…The structure of Equilin 2, was determined by Sawicki et al (1999b), who demonstrated that the presence of the unsaturated C7-C8 bond in the B ring rotates the C and D rings of the steroid such that the 17-keto oxygen atom, O17, is translated by 0.73 Å with respect to the analogous oxygen atom of 3 when an overlay of the two structures was performed based on the superposition of the A rings. The translation of the oxygen atom was implicated in the increased anti-human estrogenic 17-hydroxysteroid dehydrogenase 1 (17-HSD1) inhibitory behaviour of 2 with respect to 17-estone 3 (Sawicki et al, 1999a). The impact of the inhibitory behaviour of 2 is that it causes a reduction of the active estrogen, 17-estradiol, which is present in elevated concentrations in human breast tumour tissues and responsible for the accelerated growth of the tumour tissue.…”

Design and validation of specific inhibitors of 17 -hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis

“…Later, equilin (Fig. 3e), an equine oestrogen that is a major component of Premarin, used in hormone replacement therapy, was also shown to inhibit 17b-HSD1 (IC 50 !1 mM), and the crystal structure of the 17b-HSD1 homodimer in a ternary complex with NADP C and equilin was solved (Sawicki et al 1999). Equilin binds at the substrate binding site, with the substrate entry loop in a closed conformation.…”

Comparative investigation of thein vitroinhibitory potencies of 13-epimeric estrones and D-secoestrones towards 17β-hydroxysteroid dehydrogenase type 1

“…The surface of the tunnel is complementary to the C 18 steroidal scaffold and ensures selectivity towards estrogenic substrates 26 . At the C-terminal recognition end, hydrophilic amino acids form hydrogen-bonds to the 3-hydroxy group of the substrate 27,28 .…”