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Pseudo-symmetry of C19 steroids, alternative binding orientations, and multispecificity in human estrogenic 17ß-hydroxysteroid dehydrogenase¹

Oncology and Molecular Endocrinology Research Center, CHUL Research Center and Laval University, Québec, Canada, G1V 4G2

³Correspondence: Molecular Endocrinology and Oncology Research Center, 2705 Blvd. Laurier, Québec, Québec, Canada, G1V 4G2. E-mail: sxlin{at}crchul.ulaval.ca

SPECIFIC AIM

The original aim of the study was to understand the molecular basis for steroid recognition and discrimination by human estrogenic 17ß-hydroxysteroid dehydrogenase (17ß-HSD1). We demonstrate by enzyme kinetics and X-ray crystallography that 17ß-HSD1 can bind and catalyze C19 steroids in both normal and reverse binding orientations. Our findings suggest a possible role of 17ß-HSD1 in reducing DHT levels in peripheral intracrine tissues, especially in the human mammary gland where 17ß-HSD1 and DHT are both present.

PRINCIPAL FINDINGS

1. Simultaneous 3ß reduction and 17ß oxidation of ¹⁴C DHT by human 17ß-HSD1 resulting in inactivation of ¹⁴C DHT
We have analyzed the reduction and oxidation of dihydrotestosterone (DHT) by human 17ß-HSD1. When DHT is reduced with NADPH, 3ß-androstanediol (3ß-diol) is produced, demonstrating that 17ß-HSD1 possesses some 3ß-HSD activity; the oxidized product in position 17 (androstanedione [A-dione]) appears simultaneously with the reduced product in position 3 (3ß-diol), although in much lower quantities. These results demonstrate that the oxidized form of the cofactor NADP is used for a second reaction in which a new molecule of DHT is oxidized to yield A-dione. A similar phenomenon is observed for the oxidation reaction in the presence of NADP where the reduced product (3ß-diol) appears subsequently to the oxidized product (A-dione). These results confirm that DHT can bind to 17ß-HSD1 in the direct or the reverse orientation.

2. Reduction and oxidation of DHT by 17B-HSD1 expressed in stably transformed HEK-293 cells
To verify whether the reduction of DHT in 3ß-diol could occur in intact cells, experiments were conducted in HEK-293 cells stably transformed with human 17ß-HSD1 (Fig. 1 ). The negative control experiment conducted on the nontransformed HEK-293 cells revealed no enzymatic activity on estrone, showing that no 17ß-HSD1 activity was initially present in the nontransformed cells. The positive control experiment showed that estrone was converted by the transformed cells, consistent with 17ß-HSD1 activity. Finally, the reduction of DHT by 17ß-HSD1 in the presence of NADPH is observed in intact HEK 293 transformed cells. Similar to what is observed for the purified enzyme, the oxidation product appears simultaneously with the reduction product, suggesting the physiological significance of our finding.

View larger version (118K): [in this window] [in a new window]   Figure 1. Simultaneous reduction and oxidation of DHT by HEK-293 cells stably transformed with 17ß-HSD1. The reactions were performed at 37°C in MEM medium containing 10% v/v calf fetal serum. The 14C estrone radioactive source is shown in lane 1. Lane 2 is a negative control showing that 1 x 106 wild-type HEK-293 cells do not transform estrone into estradiol in the presence of 1 µM estrone and NADPH, demonstrating that HEK 293 cells do not have 17ß-HSD activity. Lane 3 is a positive control showing that 1 x 106 HEK 293 cells stably transformed with 17ß-HSD1 in the presence of 1 µM estrone and NADPH demonstrates 17ß-HSD1 activity. Lane 4: 1 x 106 HEK 293 cells stably transformed with 17ß-HSD1 in the presence of 1 µM DHT and NADPH, demonstrating that 17ß-HSD1 is able to reduce DHT in its 3-position and simultaneously oxidize DHT in its 17-position in intact cells. As a reference, 14C DHT is represented in lane 5. The migration system was 4:1 toluene:acetone and results were analyzed using a STORM device.

3. Structure of the 17ß-HSD1-testosterone binary complex: reverse binding orientation of the steroid
We have solved the crystal structure of a 17ß-HSD1-C19 steroid binary complex using testosterone as an example. The data between 13.9 Å and 1.54 Å were used in the refinement, yielding a crystallographic R factor of 19.6% and a free R factor of 20.8% based on a subset of 5% of the reflections. The final model includes residues 1–190 and 198–285, one testosterone molecule, one glycerol molecule, and 225 water molecules.

The high quality of the electron density map permitted the orientation of testosterone unambiguously in the binding pocket (Fig. 2 ). Similar to estradiol (E₂), testosterone binds in the narrow hydrophobic tunnel of 17ß-HSD1 with a high degree of complementarity. Surprisingly, testosterone is bound in an alternative orientation to the enzyme compared with the binding mode described for E₂ and other published 17ß-HSD1 steroid complexes. To roughly reach the position observed for testosterone, estradiol needs to rotate twice, first undergoing a rotation of 160° around the axis perpendicular to its ß-face and then a second rotation of 20° around its long axis (O3-O17).

View larger version (56K): [in this window] [in a new window]   Figure 2. Stereoview of the 2mFo-dFc electron density map around the testosterone and the active site residues in 17ß-HSD1-testosterone complex. The map computed with 1.54 Å resolution data is contoured at the 1.8 level. The figure was produced using Molray.

The 17ß-hydroxyl group located on the D-ring of testosterone binds to the substrate recognition end of the protein. It occupies almost the same position as the one observed for the phenolic hydroxyl of estradiol, with a shift of only 0.65Å, which permits formation of a strong bifurcated hydrogen bond between the 17ß-hydroxyl group of testosterone and residues His221 and Glu282 of the enzyme. However, such a modification in orientation results in a big shift for the A-ring of testosterone compared with the D-ring of E₂.

CONCLUSIONS AND SIGNIFICANCE

We have shown for the first time that 17ß-HSD1 can bind C19 steroids in both normal and reverse orientations, bringing new information on interactions existing between the enzyme and its ligands. These alternative binding modes can be explained by the pseudo-symmetry of C19 steroids. Although C19 steroids are not perfectly symmetric molecules, they are sufficiently symmetric to effect a rotation causing the 3-end to be in the usual position of the 17-end. The interchange of the A and B rings with the C and D rings does not result in a very different final position in terms of overall topology from the initial position. The fact that the pseudo-symmetry of C19 steroids allows alternative binding orientations is supported by the similar K_m measured for the 3ß reduction and the 17ß oxidation of DHT. On the other hand, estrogens do not show the same level of symmetry as androgens, because they lack the C19-methyl group and possess a planar/aromatic A-ring. These features were found to be important in order to recognize and correctly orient estradiol in the active site. The fact that alternative binding modes were not observed for estradiol but were for C19 steroids suggests that alternative binding is related to the mechanism that permits discrimination of estrogens from androgens. Previous studies have suggested that the residue Leu149 plays an important role in the discrimination between estrogens and androgens. We have shown that when testosterone is positioned in the same binding mode as estradiol, a steric clash between the 19-methyl group and the residue Leu149 is observed. We thus propose that this steric hindrance may compel testosterone to bind in a reverse binding mode.

Alternative binding mode could turn out to be important physiologically. Compared with the k_cat value of the 17ß reduction of estrone, 17ß-HSD1 shows a poor k_cat value for the 3ß reduction of DHT. Nevertheless, the activity of 17ß-HSD1 for inactivation of DHT into 3ß-diol is comparable or even better than the reported activity of other androgen-converting enzymes such as human 3-HSD3 and 17ß-HSD type 5. Moreover, experiments conducted on HEK-293 transformed cells showed that the 3ß reduction of DHT did occur in cells and could occur in vivo. The simultaneous 3ß reduction and 17ß oxidation of DHT by 17ß-HSD1 are additive in the inactivation of the most potent androgen. Therefore, it is possible that 17ß-HSD1 can participate in reducing DHT levels in the peripheral intracrine tissues and act as a modulator of the occupancy of the androgen receptor in these tissues, especially in the human mammary gland where both 17ß-HSD1 and DHT are present.

Future investigations on other enzymes able to distinguish between C18 and C19 steroids will reveal whether reverse binding orientation is the result of a more general discrimination mechanism or if it is specific to human 17ß-HSD1. We speculate that alternative binding orientations may be a more general mechanism and could be found in other steroid-converting enzymes and receptors.

View larger version (26K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0397fje; to cite this article, use FASEB J. (December 18, 2002) 10.1096/fj.02-0397fje

² These two authors contributed equally to the work.

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