ABSTRACT Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the biosynthesis of the most potent natural estrogen 17β-estradiol (E2) from estrone (E1) in the ovary and peripheral tissues, playing a pivotal role in the progression of estrogen-dependent diseases. N-n-Butyl-N-methyl-ll-(16'α-chloro-3',17'β-dihydroxyestra-1',3',5'(10')-trien-7'α-yl)undecanamide (EM-139) was previously described as a dual-site inhibitor that can inhibit 17β-HSD1 transforming E1 into E2 and also inhibit estrogen receptor. In the present report, we describe the co-crystallization of EM-139 with 17β-HSD1 as well as the analysis of the three-dimensional structure of the enzyme/inhibitor complex. The crystal is grown under similar condition as native crystals, whereas the space group is changed to I121 never observed in other 17β-HSD1 crystals before. The steroidal moiety of the bound EM-139 molecule has shown a binding pattern similar to E2 in the E2 binary complex. The O-3 of the inhibitor develops hydrogen bonds with residues His221 and Glu282, whereas the O-17 makes hydrogen bonds with Ser142 and Tyr155. The bulky 7α-alkyl moiety of the inhibitor, which is essential for its anti-estrogenic activity but cannot be defined in the electron density, may compromise the inhibitory effect of EM-139 to 17β-HSD1. Moreover, the 16α-Cl atom shows no obvious interaction with surrounding residues. The atomic level understanding of the inhibitory mechanism of EM-139 provides important information for the inhibitor design of 17β-HSD1, which will facilitate future development of more potent and selective inhibitors of the enzyme for therapeutic purposes.
Seventeen β-hydroxysteroid dehydrogenase type 1 (17β-HSD1, EC. 126.96.36.199) catalyzes the NAD(P)H dependent conversion of estrone (E1) to the most potent estrogen, 17β-estradiol (E2) . E2 is well known to play a crucial role in the progression and development of several estrogen-dependent diseases (EDD). Increased E2 levels as well as up-regulated 17β-HSD1 expression indicate the involvement of the enzyme in EDDs, such as breast cancer , endometrial cancer , endometriosis , and ovarian cancer . Moreover, patients with tumors that have high mRNA levels of 17β-HSD1 have significantly shortened disease-free and overall survival . Therefore, blocking the production of E2 through the specific inhibition of 17β-HSD1 activity is considered to be of therapeutic benefit in the treatment of EDDs.
Over the past decades, major efforts from many different laboratories have been devoted to developing highly selective inhibitors of the key steroidogenic enzyme 17β-HSD1, yielding several lead compounds with significant inhibitory activity . However, due to the lack of specificity, especially for the presence of undesired estrogenic activity, no inhibitor has yet reached the stage of clinical trials . N-n-Butyl-N-methyl-ll-(16'α-chloro-3',17'β-dihydroxyes-tra-1',3',5'(10')-trien-7'α-yl) undecanamide (EM-139) is a 7α-alkyl, 16α-halo estradiol derivative which was first synthesized as a pure antiestrogen (Figure 1) . Following experiments demonstrated its inhibitory effect on 17β-HSD1 activity with a Ki of 6 μM . Thus the compound was defined as a dual-site inhibitor which possesses inhibitory effect on estrogen receptor and on the estrogen formation . Although this compound was proven to be a non-selective inhibitor of the 17β-HSD family members , study of the EM-139/17β-HSD1 complex structure should help us to better understand the inhibitory mechanism of the dual-site inhibitor, thus facilitating further inhibitor design of the enzyme.
Previously, we have reported the crystallization of the 17β-HSD1/EM-139 complex using both co-crystallization and soaking methods . The crystals obtained were isomorphous to the native crystals with a monoclinic space group C2 . After careful analysis of the structures, the inhibitor couldn’t be identified at the binding site of the enzyme due to poor electron density. In the present study, we optimized the co-crystallization procedure and successfully obtained complex crystals with a unique space group never observed in 17β-HSD1 complexes before. The clear electron density at the binding site indicated the presence of the dual-site inhibitor in the enzyme complex.
2. Materials and Methods
2.1. Protein Preparation and Co-Crystallization
The 17β-HSD1 enzyme was expressed in Sf9 insect cells and purified as described previously . After the measurement of specific activity , the purified enzyme was concentrated to a final concentration of 15 mg/ml in the presence of 0.06% β-octyl glucoside (β-OG), and then subjected to a buffer change procedure via centricon (Emdmillipore, USA) to saturate the enzyme with the inhibitor EM-139. The co-crystallization experiment was carried out using the vapor diffusion method at room temperature. Crystals were obtained under conditions containing 22% - 26% (w/v) polyethyleneglycol (PEG) 4000, 0.15 M magnesium chloride, and 0.1 M HEPES buffered to pH 7.5.
2.2. Data Collection and Structure Determination
The X-ray diffraction data of the 17β-HSD1-EM-139 crystals were collected at 100 K using synchrotron radiation at Advanced Photon Source (APS) beamline 31-LRL-CAT (Chicago, USA) equipped with a MAR CCD 165 mm detector at a wavelength of 0.9793 Å. The dataset was indexed and intergraded using MOSFLM , and scaled with SCALA from the CCP4 suite . The structure was solved by molecular replacement with Molrep using a reported 17β-HSD1 coordinate (PDB code 1JTV) as search model. The initial model was subjected to multiple rounds of auto-refinement using Refmac and manual rebuild using Coot . Missing portions of the models, inhibitor EM-139, glycerol, polyethylene glycol, and water molecules were progressively added with great caution during the refinement procedure. The final model was verified with PROCHECK . Molecular graphics were presented using the Pymol software (version 2.0 Schrödinger, LLC).
Crystal utilized in this study belonged to space group I121 and each asymmetric unit contained a dimer, which is known to be the functional unit of the enzyme . The complex structure was refined at 2 Å with good stereochemistries , and the quality of the final model can be assessed in Table 1. Similar to most previously reported 17β-HSD1 structures, the highly flexible βFαG’-loop (amino acids Phe192 to Leu197) as well as the C-terminal end of the protein (amino acids 286 to 327) cannot be defined in the electron density (Figure 2) .
In the binary complex structure, EM-139 has definable electron density in the A subunit of the dimeric enzyme. However, the ligand density in the B subunit is poorly defined, similar to previously described complex with equilin . Accordingly the ligand was not included in the B subunit of the final model. Even for the A subunit, only the steroid moiety of EM-139 can be defined but with a high average B-factor (97.5 Å2), whereas the 7α-alkyl side chain of the inhibitor cannot be defined in the electron density (Figure 3). This high flexibility of the inhibitor is in accordance with its relatively low affinity for the enzyme .
The space group of 17β-HSD1 crystals can be affected by the presence of cations in the crystallization conditions . The space group of crystals obtained in the
Table 1. Data collection and refinement statistics.
a. Data statistics for the outer shell are given in parentheses. b.The redundancy-independent Rmerge/Rsym,
. d. Rfree = the cross-validation R factor for 5% of reflections against which the model was not refined. e. Calculated with PROCHECK.
presence of Mg2+ and Mn2+ belong to C2, whereas crystals grown under conditions with Li+ and Na+ had a space group of P212121 . Despite the presence of Mg2+, the space group of the co-crystallized EM-139 complex crystals has been changed to I121, not observed in any other reported 17β-HSD1 structures. The change in space group may be due to the long alkyl side chain at the C7 of EM-139, which may affect the packing during crystal growth.
When the EM-139 binary and E2 binary (PDB ID 1IOL ) complexes as
Figure 1. Structure of dual-site inhibitor N-n-Butyl-N-methyl-ll-(16'α-chloro-3',17'β-dihydroxyestra-1',3',5'(10')-trien-7'α-yl)undecanamide (EM-139).
Figure 2. Stereo representation of the overall structure of A subunit of 17β-HSD1-EM-139 complex. The protein molecule is shown in cartoon and colored in pink. The bound EM-139 molecule is depicted as stick and colored in blue. The N-terminus and the C-terminus of the protein molecule are indicated. Segment of residues 190-197, which unable to be defined in the electron density, is represented as dash line.
well as the apo structure of 17β-HSD1 (PDB ID 1BHS ) are superimposed, a similar conformation is observed at the steroid binding site of the enzyme (Figure 4(a)). The root-mean-square deviation (RMSD) for all paired Cα atoms obtained between EM-139 complex and apo structure is 0.456 Å, similar to the value obtained between EM-139 and E2 complexes (0.508 Å). It is worth mentioning that the position observed for the steroidal moiety of EM-139 has roughly 9˚ rotation around the axis at the C-3 atom and perpendicular to its β-face, when compared with the position of E2. This leads to the shifting of the O-17 by 1.4 Å as compared with the position of its counterpart in the E2 complex (Figure 4(b) and Figure 4(c)). As a result, the bifurcated hydrogen bonds between the O-17 of EM-139 with Ser142 and Tyr155 (3.5 and 3.2 Å, respectively) are established, although the bond distances differ from their counterparts observed in the E2 complex (3.1 and 3.5 Å, respectively) . Moreover, the bifurcated hydrogen bond between the 3-hydroxyl group of EM-139 with His221 and
Figure 3. Front and side views of the electron density of EM-139 in the 17β-HSD1-EM-139 complex structure. EM-139 (ligand ID EM9) was shown in the omit Fo-Fc and 2Fo-Fc electron density. 2Fo-Fc map draw in gray and contoured at 0.8σ; Fo-Fc map draw in green and contoured at 1.5σ. The occupancy of the inhibitor was refined to 1. No significant negative density features were present in the region of binding site.
Glu282 (3.2 and 3.5 Å, respectively) at the recognition end of the steroid binding cleft is conserved. Although much weaker as compared to their counterparts in the E2 complex (3.1 and 2.7 Å, respectively) , these hydrogen bonds are essential for stabilizing the inhibitor in the steroid binding cavity together with the hydrogen bonds at the O-3 of EM-139.
The 7α-alkyl moiety of EM-139 is facing toward the outside of the steroid binding cavity which is apparently accommodated by the βFαG’-loop. However, both the 7α-alkyl side chain of the inhibitor and the βFαG’-loop of the enzyme are unable to be defined by electron density due to their high degree of flexibility. This bulky 7α-alkyl side chain is essential for the inhibitor to possess anti-estrogenic activity . It is also safe to conclude that the α conformation of the C-7 is essential for this compound to be able to bind with 17β-HSD1. Similar results can also be observed at the C-16 of the inhibitor where a 16β halogen atom may have steric hindrance with Tyr155. However, no obvious interaction is observed between the 16α-Cl atom and surrounding residues (Figure 4(b) and Figure 4(c)).
The present work was aimed at investigating the molecular basis of the inhibitory mechanism of the dual-site inhibitor EM-139 in 17β-HSD1. We successfully co-crystallized and solved the crystal structure of 17β-HSD1 in complex with the inhibitor. Through comparative analyses of EM-139 binary complexes and previously reported E2 binary complex as well as the apo structure, we observed a similar binding pattern of the inhibitor to the enzyme. The bifurcated hydrogen bonds between the O-3 of the inhibitor and the recognition end (His221 and Glu282) of the binding site as well as the O-17 of the inhibitor and the catalytic
Figure 4. Superposition of A subunit of EM-139 (EM9) binary complex (pink) and E2 binary complex (cyan) along with 17β-HSD1 apo structure (orange), showing the steroid ligand binding sites. (a) General view of the active sites within the A subunit of EM-139 and E2 complex structures as well as the apo structure; (b) Top and (c) side view of the steroid binding sites in the superposed structures. Residues Ser142, Leu149, Tyr155, His221, and Glu282 are labeled and shown in sticks. Hydrogen bonds between EM-139 and surrounding residues are drawn in green dash lines and labeled. Chloride atom is colored in green.
end (Ser142 and Tyr155) of the binding site are critical in stabilizing the bound inhibitor molecule. However, the introduction of a bulky side chain at the C-7 of the steroid core, which contributes to the anti-estrogenic activity of the dual-site inhibitor, may negatively affect the binding of inhibitor to 17β-HSD1. These results will contribute to the design of more potent and selective inhibitors of 17β-HSD1 for clinical purposes.
We thank Dre. M. Zhou for her technical support in enzyme purification and crystal growth. We acknowledge the use of beamline 31-LRL-CAT at the Advanced Photon Source, Argonne National Laboratory, USA.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.
Cite this paper
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