Steroid recognition and regulation of hormone action: crystal structure of testosterone and NADP+ bound to 3α-hydroxysteroid/dihydrodiol dehydrogenase

Abstract Background: Mammalian 3 α -hydroxysteroid dehydrogenases (3 α -HSDs) modulate the activities of steroid hormones by reversibly reducing their C3 ketone groups. In steroid target tissues, 3 α -HSDs act on 5 α -dihydrotestosterone, a potent male sex hormone (androgen) implicated in benign prostate hyperplasia and prostate cancer. Rat liver 3 α -HSD belongs to the aldo-keto reductase (AKR) superfamily and provides a model for mammalian 3 α -, 17 β -  and 20 α -HSDs, which share >65% sequence identity. The determination of the structure of 3 α -HSD in complex with NADP + and testosterone (a competitive inhibitor) will help to further our understanding of steroid recognition and hormone regulation by mammalian HSDs. Results: We have determined the 2.5 A resolution crystal structure of recombinant rat liver 3 α -HSD complexed with NADP + and testosterone. The structure provides the first picture of an HSD ternary complex in the AKR superfamily, and is the only structure to date of testosterone bound to a protein. It reveals that the C3 ketone in testosterone, corresponding to the reactive group in a substrate, is poised above the nicotinamide ring which is involved in hydride transfer. In addition, the C3 ketone forms hydrogen bonds with two active-site residues implicated in catalysis (Tyr55 and His117). Conclusions: The active-site arrangement observed in the 3 α -HSD ternary complex structure suggests that each positional-specific and stereospecific reaction catalyzed by an HSD requires a particular substrate orientation, the general features of which can be predicted. 3 α -HSDs are likely to bind substrates in a similar manner to the way in which testosterone is bound in the ternary complex, that is with the A ring of the steroid substrate in the active site and the β face towards the nicotinamide ring to facilitate hydride transfer. In contrast, we predict that 17 β -HSDs will bind substrates with the D ring of the steroid in the active site and with the α face towards the nicotinamide ring. The ability to bind substrates in only one or a few orientations could determine the positional-specificity and stereospecificity of each HSD. Residues lining the steroid-binding cavities are highly variable and may select these different orientations.