Dao-Wei Zhu

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altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished).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.

Crystal growth of human estrogenic 17β-hydroxysteroid dehydrogenase

Estrogenic 17beta-hydroxysteroid dehydrogenase from human placenta, an enzyme of low solubility, has been crystallized in the complex form with its cofactor NADP(+). These are the first crystals with X-ray diffraction quality for structure analysis from any human steroid-converting enzyme. The crystals were grown by vapor diffusion in the presence of 0.06% beta-octylglucoside, using polyethylene glycol 4000 as the precipitant (27-28%) and one of several different salts at pH 7.5 and room temperature. Crystals grown with magnesium chloride diffract up to 2.4 A. The most important steps leading to the rapid success of the crystallization of this labile enzyme were the following: preparation of a highly active and homogeneous enzyme protein using a rapid procedure; the choice of a suitable enzyme buffer system and a detergent favorable to maintaining high activity and solubility for the enzyme; and a combined screening procedure. The present study could be useful for the successful crystal growth of other hydrophobic or membrane-bound proteins.

Primary Sequence and Refined Tertiary Structure of Pseudomonas fluorescens Holo Azurin at 2.05 A

This paper reports the primary sequence and refined crystal structure of Pseudomonas fluorescens holoazurin. The crystal structure has been determined by molecular replacement on the basis of the molecular model of azurin from Alcaligenes denitrificans, and refined by the method of molecular dynamics simulation and energy-restrained least-squares methods. P. fluorescens was crystallized in the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions a = 31.95, b = 43.78, c = 78.81 A. The asymmetric unit is composed of only one molecule. The final R value is 16.7% for 6691 reflections to a resolution of 2.05 A. This azurin structure shows some interesting features at His35 and His83. Part of the main chain of strand 3 including His35 O are involved in the contact between two symmetrically related molecules. P. fluorescens is also compared with the other azurin structures in terms of primary sequence, crystal packing, solvent structure and Cu-site geometry. The difference in fluorescence decay behavior of two holoazurins from P. fluorescens and P. aeruginosa and the correlation between the fluorescence quenching and electron transfer are discussed.

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.

Crystallization of the Membrane-Associated Annexin B1: Roles of Additive Screen, Dynamic Light Scattering, and Bioactivity Assay

Annexin B1 (AnxB1) is a calcium-dependent phospholipid binding protein from Taenia solium cysticercus and has been reported to possess anticoagulant activity, to inhibit phospholipase A2, and to regulate membrane transport. Native AnxB1 and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. The results of dynamic light scattering analysis showed that Hepes buffer combined with low concentration salts (NaCl or CaCl2) was beneficial for preventing aggregation and for AnxB1 stabilization in the storage. After the additive screen, crystals have been yielded in the presence of guanidine hydrochloride (Gn-HCl). We determined that a low concentration of Gn-HCl significantly delayed clotting time and increased anticoagulant activity. Analysis of the crystal showed that in the presence of Gn-HCl, AnxB1 crystallizes in orthorhombic space group, which is modified from the cubic space group for crystals grown in the absence of Gn-HCl. A high quality data set (at 1.9 A) has been co...