Human 20α–Hydroxysteroid Dehydrogenase: Crystallographic and Site-directed Mutagenesis Studies Lead to the Identification of an Alternative Binding Site for C21-steroids

Human 20α–hydroxysteroid dehydrogenase (h20α–HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20α–hydroxy-progesterone (20α–OHProg). Although h20α–HSD shares 98% sequence identity with human type 3 3α–HSD (h3α–HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20α–HSD in a ternary complex with NADP+ and 20α–OHProg at 1.59 Å resolution. The steroid is stabilized by numerous hydrophobic interactions and a hydrogen bond between its O20 and the Nε atom of His222. This new interaction prevents the formation of a hydrogen bond with the cofactor, as seen in h3α–HSD3 ternary complexes. By combining structural, direct mutagenesis and kinetic studies, we found that the H222I substitution decreases the Km value for the cofactor 95-fold. With these results, we hypothesize that the rotation of the lateral chain of His222 could be a mediating step between the transformation of Prog and the release of the cofactor. Moreover, crystal structure analysis and direct mutagenesis experiments lead us to identify a new residue involved in the binding of Prog. Indeed, the R304L substitution leads to a 65-fold decrease in the Km value for Prog reduction. We thus propose that Prog is maintained in a new steroid-binding site composed mainly of residues found in the carboxy-terminal region of the protein.