Homology modeling meets site-directed mutagenesis: An ideal combination to elucidate the topology of 17β-HSD2

Highlights

• First homology model of 17β-HSD2.

• 17β-HSD2 homology model quality validated by side-directed mutagenesis.

• Elucidation of the role of specific amino acids.

Abstract

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the conversion of highly active estrogens and androgens into their less active forms using NAD⁺ as cofactor. Substrate and cofactor specificities of 17β-HSD2 have been reported and potent 17β-HSD2 inhibitors have been discovered in a ligand-based approach. However, the molecular basis and the amino acids involved in the enzymatic functionality are poorly understood, as no crystal structure of the membrane-associated 17β-HSD2 exists. The functional properties of only few amino acids are known. The lack of topological information impedes structure-based drug design studies and limits the design of biochemical experiments. The aim of this work was the determination of the 17β-HSD2 topology. For this, the first homology model of 17β-HSD2 in complex with NAD⁺ and 17β-estradiol was built, using a multi-fragment "patchwork" approach. To confirm the quality of the model, fifteen selected amino acids were exchanged one by one using site directed mutagenesis. The mutants’ functional behavior demonstrated that the generated model was of very good quality and allowed the identification of several key amino acids involved in either ligand or internal structure stabilization. The final model is an optimal basis for further experiments like, for example, lead optimization.

Introduction

Knowledge of the 3D-structure and binding site topology of an enzyme is an essential prerequisite for an effective structure-based drug design and the facilitated design of biochemical experiments.

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2, EC1.1.1.51) [1,2] is a homodimeric enzyme, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. The protein is localized in the endoplasmic reticulum, and its mRNA is widely expressed in various human peripheral tissues [[3], [4], [5], [6], [7]]. Using NAD⁺ as cofactor, 17β-HSD2 has a predominant 17β-oxidative activity catalyzing the conversion of highly active 17β-hydroxy forms of estrogens and androgens (i.e., estradiol (E2), testosterone (T), and dihydrotestosterone) into their less active keto forms (i.e., estrone (E1), Δ⁴-androstene-3,17-dione (Δ4-Ae), and 5α-androstanedione, respectively). Furthermore, the enzyme also possesses a 20α- and a weak 3β-oxidase activity [8].

Several research groups successfully characterized the substrate and cofactor specificities of 17β-HSD2 [9] and developed potent 17β-HSD2 inhibitors using ligand-based strategies [10]. In addition, Bagi et al. [11] have shown by in vivo experiments using a monkey model, that 17β-HSD2 inhibition leads to increasing E2 concentration in the bones and thus might be useful for the treatment of osteoporosis. This result was confirmed recently in a mouse and a rat model [12, 13].

However, only for some of all the 17β-HSD2 amino acid residues the role in enzyme functionality is evident: Ser219, Tyr232, and Lys236, for example, constitute the SDR-conserved catalytic triad [14], while Glu116 was reported as not being essential for the catalytic activity of 17β-HSD2 [9].

As 17β-HSD2 is strongly associated with the membrane of the endoplasmic reticulum [2] through its N-terminal end [15], purification of the full-length enzyme is very difficult and up to now, only one research group was able to successfully purify the full-length 17β-HSD2 in its enzymatically active form [2]. However, the 3D-crystal structure of 17β-HSD2 still remains unknown. This impedes structure-based drug design studies, limits a precise design of biochemical experiments, and prevents a structure comparison between 17β-HSD2 and already crystallized 17β-HSDs, an essential step for selectivity investigations.

Whereas Engeli et al. recently used a homology model approach to explain the mutation-based loss of function of 17β-HSD3 [16,17], the aim of the study here was to generate a good quality homology model of 17β-HSD2 to get insight in the enzyme's topology. For this, a broad set of 17β-HSD2 homology models was generated using automatized and manual multi-template approaches. Representative 17β-HSD2-NAD⁺-E2 ternary models were evaluated and a final refined 17β-HSD2 model could be obtained. Based on this model, functionally essential residues in the cofactor and substrate binding sites were identified and their role in the structure confirmed using mutant proteins generated by site-directed mutagenesis. The comparison of the biological activity of the wild type enzyme with the one of the mutants validated the very good quality of the 17β-HSD2 homology model.