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 17β-estradiol (E₂), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), which catalyzes the reduction of inactive estrone (E₁) to the active 17β-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E₂ in breast tumor tissues. We present here the structure of the ternary complex of 17β-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 17β-HSD1-catalyzed reduction of E₁ to E₂ 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 E₂ in the 17β-HSD1–E₂ 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 17β-HSD1 is the basis for inhibition of E₁-to-E₂ reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E₂ levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.