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...

CD69 is a widely expressed type II transmembrane glycoprotein related to the C-type animal lectins that exhibits regulated expression on a variety of cells of the hematopoietic lineage, including neutrophils, monocytes, T cells, B cells, natural killer (NK) cells, and platelets. Activation of T lymphocytes results in the induced expression of CD69 at the cell surface. In addition, cross-linking of CD69 by specific antibodies leads to the activation of cells bearing this receptor and to the induction of effector functions. However, the physiological ligand of CD69 is unknown. We report here the X-ray crystal structure of the extracellular C-type lectin-like domain (CTLD) of human CD69 at 2.27 A resolution. Recombinant CD69 was expressed in bacterial inclusion bodies and folded in vitro. The protein, which exists as a disulfide-linked homodimer on the cell surface, crystallizes as a symmetrical dimer, similar to those formed by the related NK cell receptors Ly49A and CD94. The structure reveals conservation of the C-type lectin-like fold, including preservation of the two alpha-helical regions found in Ly49A and mannose-binding protein (MBP). However, only one of the nine residues coordinated to Ca(2+) in MBP is conserved in CD69 and no bound Ca(2+) is evident in the crystal structure. Surprisingly, electron density suggestive of a puckered six-membered ring was discovered at a site structurally analogous to the ligand-binding sites of MBP and Ly49A. This sugar-like density may represent, or mimic, part of the natural ligand recognized by CD69.