Two non-reactive ternary complexes of estrogenic 17b- hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis Wei Qiu, Dao-Wei Zhu, Arezki Azzi, Robert L. Campbell, Heng Qi, Donald Poirier, Sheng-Xiang Lin* MRC Group in Molecular Endocrinology, CHUL Research Center and Laval University, Quebec, Qc, G1V 4G2, Canada Received 15 October 1998; accepted 18 December 1998 Abstract Human estrogenic 17b-hydroxysteroid dehydrogenase (17b-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17b-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41). To further study the transition state, two non-reactive ternary complexes, 17b-HSD1±EM519-NADP + and 17b-HSD1± EM553-NADP + were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have dierent unit cells, with a=155.59 A Ê , b=42.82 A Ê , c=121.15 A Ê , b=128.58 for 17b-HSD1±EM519- NADP + , and a=124.01 A Ê , b=45.16 A Ê , c=61.40 A Ê , b=99.28 for 17b-HSD1±EM553-NADP + , respectively. Our preliminary results revealed that the inhibitors interact dierently with the enzyme than do the natural substrates. # 1999 Elsevier Science Ltd. All rights reserved. 1. Introduction 17b-hydroxysteroid dehydrogenases (17b-HSDs) constitute a family of isoenzymes that can be found in all classical steroidogenic tissues and most peripheral tissues [1]. They catalyze the oxidation and reduction of steroid hormones. Among this family, 17b-HSD type 1 has been well studied. In breast tumours, 17b- estradiol (E 2 ) was found in signi®cantly higher concen- tration than in normal breast tissues [2,3]. Human 17b-HSD1, which is responsible for the formation of the potent estrogen estradiol (E 2 ) from estrone (E 1 ) [4], has high activity in malignant breast tissues. Thus, this enzyme is an important target for inhibitor design and breast cancer therapy. After the determination of the ®rst 3-D crystallo- graphic structure for 17b-HSD1 [5], the structure± function relationship of the enzyme was much better understood, which in turn stimulated further studies of the enzyme structure. The crystallographic studies on this enzyme have been focusing on two main aspects. One aspect is the determination of structural details using site-directed mutagenesis. For example, the im- portance of His 221 to the catalytic activity of 17b- HSD1 was demonstrated by the kinetic study of the mutated enzyme [6]. Then this result was proved by the crystal structures of H221L 17b-HSD1 mutant/ NADP + and estradiol complexes, the H221L mutant/ NAD + , and the H221Q mutant/estradiol complexes [7]. The other and more direct aspect consists in study- Journal of Steroid Biochemistry and Molecular Biology 68 (1999) 239±244 0960-0760/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0960-0760(99)00036-9 * Corresponding author. Tel.: +1-418-6542296; fax: +1-418- 6542761. E-mail address: sxlin@crchul.ulaval.ca (S.X. Lin)