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Comparison of crystal structures of human type 3 3-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen

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

(RECEIVED January 11, 2005; FINAL REVISION March 17, 2005; ACCEPTED March 18, 2005)

The aldo-keto reductase (AKR) human type 3 3-hydroxysteroid dehydrogenase (h3–HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5-dihydrotestosterone (5-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3–HSD3-NADP(H) at 1.9 Å resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3–HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of themobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 ų in the presence of testosterone/acetate to 704 ų in the acetate/citratecomplex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg₃₀₁ and Arg₃₀₄, localized near the steroid-binding cavity, significantly affects the 3–HSD activity of this enzyme toward 5-DHT and completely abolishes its 17–HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.

Keywords: aldo-keto reductase; hydroxysteroid dehydrogenase; crystal structure; induced-fit mechanism

Abbreviations: h3–HSD3, human 3-hydroxysteroid dehydrogenase type 3 • h20–HSD, human 20-hydroxysteroid dehydrogenase • h3– HSD1, human 3-hydroxysteroid dehydrogenase type 1 • 17–HSD, 17-hydroxysteroid dehydrogenase • NADP(H), reduce form of nicotinamide adenine dinucleotide phosphate • GST, glutathione sulfotransferase • PDB, Protein Data Bank • AKR, aldo-keto reductase • progesterone, 5-pregnen- 3,20-dione • 4-dione, 4-androsten-3,20-dione • PAH, polycyclic aromatic hydrocarbon • EDTA, ethylene diamine tetraacetic acid.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051353205.

Reprint requests to: Rock Breton, Centre de Recherche en Endocrinologie Moléculaire et Oncologique, Centre Hospitalier de l’Université Laval, 2705, boul. Laurier, Ste-Foy, QC, G1V 4G2, Canada; e-mail: rock.breton{at}crchul.ulaval.ca; fax: (418) 654-2761.

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