RESEARCH WITHIN THE PROTEIN CRYSTALLOGRAPHY GROUP OF THE LABORATORY OF MOLECULAR ENDOCRINOLOGY

The primary focus of our research is to determine the structural & functional roles of enzymes involved in steroid hormone biosynthesis. To achieve this we make use of X-ray crystallography, various physical and chemical means (such as kinetic), and binding and fluorescence studies. The steroid biosynthetic enzymes are of interest because of the prevalence of hormone-sensitive breast and prostate cancers. These enzymes are therefore targets for the development of specific inhibitors to be used in the treatment of these cancers.

We have grown diffraction-quality crystals of the estrogenic 17ß-HSD (17ß-HSD1). This is the first crystallization of any steroid-converting enzyme from human or mammalian sources (Zhu et al & Lin, 1993). The primary 17ß-HSD1 structure and a number of the enzyme’s complex structures have been discovered by our group (Ghosh et al. & Lin, 1995; Azzi et al. & Lin, 1996). This is a homo-dimeric enzyme (Lin et al. & Labrie, 1992) of 327 amino acids per monomer. It belongs to the short-chain alcohol dehydrogenase (SCAD) family. The SCAD enzymes are a family of single-domain alpha/beta proteins containing a nucleotide-binding fold (Rossmann fold) as well as various secondary structural elements that are involved in substrate recognition. Overall, the sequence homology among members of this family is quite low.

Six types of 17ß-HSD with different substrate and cofactor specificities and different preferences for reduction versus oxidation have been sequenced so far. 17ß-HSD1 catalyzes the reduction of the O17 oxygen of a number of steroids. Its primary substrate is estrone (converting it to estradiol), but it has some affinity for other substrates such as dehydroepiandrosterone (DHEA), androstanedione, androstenedione (a4-dione) and progesterone (at the O20 oxygen). Unlike some other dehydrogenases and reductases, 17ß-HSD1 is capable of utilizing either NAD+ or NADP+, but has a preference for NADP+. The type 2 17ß-HSD is known to play a role in the oxidation of active steroid hormones, while type 3 is known to play a role in the biosynthesis of testosterone from DHT.

Recently, the structures of 17ß-HSD1 with DHEA and dihydrotestosterone (DHT) complexes have been determined by this group. Structures of the enzyme bound to various inhibitors with and without NADP+ cofactor are also in progress. These studies will provide detailed information on substrate specificity and inhibition of the enzyme.

The study of the crystallogenesis of proteins, including crystallization under microgravity conditions, constitutes an important part of the research within our group (Zhu et al. & Lin, 1996). Several improvements in protein crystallization methods have been achieved in this laboratory. We were successful in obtaining crystal of a better quality during space flights aboard MIR and a Chinese recoverable satellite in 1996 and 1997.

We are working on the expression, rapid purification and crystallization of 17ß-HSD2, 17ß-HSD3 and 3ß-HSD, which are more hydrophobic and more tightly associated with the membrane than is 17ß-HSD1. We have also purified and are currently crystallizing 17ß-HSD5, which is not a member of the SCAD family, but rather of the aldo-keto reductases. The latter 17ß-HSD & HSD5 are a sub-family of the 8-stranded beta/alpha barrel proteins.

These projects are supported by the Medical Research Council Group in Molecular Endocrinology, the Canadian Space Agency and the Fonds FCAR.

Ghosh D, Pletnev VZ, Zhu D-W, Wawrzak Z, Duax WL, Pangborn W, Labrie F and Lin S-X (1995) "Structure of human estrogenic 17ß-hydroxysteroid dehydrogenase at 2.20Å resolution". Structure 3, 503-513.

Azzi A, Rehse PH, Zhu D-W, Campbell RL, Ghosh D, Labrie F. and Lin S-X (1996) "Crystal structure of human estrogenic 17ß-hydroxysteroid dehydrogenase complexed with 17beta-estradiol." Nature Structural Biology 3, 665-668.

Zhu, D.-W, Lee, X. & Lin, S.-X. (1994), Crystal growth of human estrogenic 17ß-hydroxysteroid dehydrogenase. Acta Crystallographica D: 550-555.

Lin, S.-X. et al & Labrie (1992), Subunit identity of the dimeric 17ß-hydroxysteroid dehydrogenase from human placenta. J. Biol. Chem. 267: 16182-16187.