Sheng-Xiang Lin

Dehydroepiandrosterone and dihydrotestosterone recognition by human estrogenic 17beta-hydroxysteroid dehydrogenase. C-18/c-19 steroid discrimination and enzyme-induced strain.

Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein. Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. TheK m of estrone is more than 1000-fold lower than that of DHEA and the K m of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17β-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 → Val variant showed a significantly decreased K m for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a β-oriented form, which approaches the transition-state conformation for DHEA reduction.

3D-structure of human estrogenic 17β-HSD1 : binding with various steroids

Human estrogenic dehydrogenase (17beta-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure function relationships of 17beta-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17beta-HSD1 in complex with different steroids. The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665-668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17beta-HSD1 in complex with dihydrotestosterone and 20alpha-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 A. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17beta-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.

The crystallogenesis of a human estradiol dehydrogenase-substrate complex

Abstract Human 17β-hydroxysteroid dehydrogenase type 1 is an important steroidogenic enzyme catalyzing the synthesis of the most active estrogen: estradiol. The enzyme is formed by two identical subunits (34.5 kDa). In this paper, we report the preparation of a stoichiometric 17β-HSD1-estradiol complex sample at a much higher concentration than the solubility of the free substrate, using a gradual concentration of the enzyme-substrate mixture starting at low concentration. The complex is successfully crystallized by vapor diffusion at pH 7.5 with polyethyleneglycol 4000 as the precipitating agent. The space group is C2 with a = 123.56 A , b = 45.21 A , c = 61.30 A and β = 99.06°. There is one monomer in the asymmetric unit and two molecules of the enzyme in a unit cell. A diffraction data set to 2.5 A has been collected to 86% completeness on native crystals. The high quality of the electronic density map of estradiol supports the full occupancy of the binding site, thus confirming the efficiency of the complex preparation. This method will also be useful in crystallizing other steroid-dehydrogenase complexes.

Rapid purification of the over-expressed membrane 3 β-hydroxysteroid dehydrogenase in the presence of detergents

Abstract Three-beta hydroxysteroid dehydrogenase / Δ 5 -Δ 4 isomerase catalyses a key step in the transformation of all 5-prognen-3β-ol and 5-androsten-3β-ol steroids into the corresponding Δ 4 -3-keto-steroids. Human type I 3β-HSD can be found in the subcellular fractions of mitochondria and microsome. A 1.5 kbp cDNA encoding human type I 3β-HSD was inserted into the transfer vector pBlueBac to form plasmid pBB / 3β-HSD. The recombinant baculovirus was obtained by co-transfection of wild type AcNPV genomic DNA and PBB / 3β-HSD in Sf9 cells, then used to infect Sf9 cells to over-express human 3β-HSD protein. The 3β-HSD sample was purified to homogeneity by a rapid procedure, consisting of an anion-exchange and an adsorbance chromatographies, based on FPLC and some detergents application. The whole process was successful with a purification rate of 90 fold and a high recovery (70%). The kinetic study showed a Vmax of 500 nmol/min · mg and a Km of 2.8 μM, being much more active than those reported.

Crystallization of human estrogenic 17β-hydroxysteroid dehydrogenase under microgravity

Abstract Human 17β-hydroxysteroid dehydrogenase has been crystallized on the ground in the complex form with NADP + and a complete data set of the crystal was primarily collected at 2.9 A [D.-W. Zhu, X. Lee, R. Breton, D. Ghosh, W. Pangborn, W.L. Duax and S.-X. Lin, J. Mol. Biol. 234 (1993) 242]. To eliminate multiseeding, formation of multicrystals and to obtain higher quality crystals, we carried out the crystallization aboard the Russian MIR space station and crystals were recovered in January, 1994. Crystals of the enzyme were formed in 9 of the total 12 sitting drops in the space mission, in the presence of NADP + or estradiol. This is a first attempt of crystallization of a membrane-associated protein under microgravity in the presence of a detergent. The space experiments showed better results in nucleation number, crystal size and morphology than the ground ones, obtaining crystals diffracting to resolutions between 2.5–2.7 A. The too early ground mixing has limited a more important improvement of the crystallization.

Human 17β-hydroxysteroid dehydrogenase-ligand complexes: crystals of different space groups with various cations and combined seeding and co-crystallization

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD 1) is responsible for the synthesis of active estrogens that stimulate the proliferation of breast cancer cells. The enzyme has been crystallized using a Mg 2+ . PEG (3500). β-octyl glucoside system [Zhu et al., J. Mol. Biol. 234 (1993) 242]. The space group of these crystals is C2. Here we report that cations can alTect 17β-HSD1 crystallization significantly. In the presence of Mn 2- instead of Mg 2+ , crystals have been obtained in the same space group with similar unit cell dimensions. In the presence of Li + and Na + instead of Mg 2+ , the space group has been changed to P2 1 2 1 2 A whole data set for a crystal of 17β-HSD1 complex with progesterone grown in the presence of Li + has been collected to 1.95 A resolution with a synchrotron source. The cell dimensions are a = 41.91 A, h = 108.21 A, c = 117.00 A. The structure has been preliminarily determined by molecular replacement. yielding important information on crystal packing in the presence of different cations. In order to further understand the structure-function relationship of 17β-HSD1 enzyme complexes with several ligands have been crystallized. As the steroids have very low aqueous solubility, we used a combined method of seeding and co-crystallization to obtain crystals of 17β-HSD l complexed with various ligands. This method provides ideal conditions for growing complex crystals, with ligands such as 20α-hydroxysteroi progesterone, testosterone and 17β-methyl-estradiol-NADP + Several complex structures have been determined with reliable electronic density of the bound ligands.