Journal of Molecular Biology
Loop Relaxation, A Mechanism that Explains the Reduced Specificity of Rabbit 20α-Hydroxysteroid Dehydrogenase, A Member of the Aldo-Keto Reductase Superfamily
Introduction
The aldo-keto reductase (AKR) superfamily encompasses more than 100 proteins with broad physiological roles. It includes a number of mammalian hydroxysteroid dehydrogenases (HSDs), including the 3α–HSDs, 20α–HSDs, and type 5 17β–HSD. Although highly homologous (more than 80% amino acid identity), all these HSDs are very selective and several of their characteristics differ considerably, including substrate specificity and diversity of activities.
AKR family members take the form of a (β/α)8 TIM barrel fold1 consisting of a cylindrical core of eight parallel β-strands, forming the β-barrel, surrounded by eight α-helices running antiparallel to the strands. The carboxy ends of the β-strands are connected to amino ends of the α-helices by loops of varying lengths forming the active site. In fact, residues that participate in substrate binding and catalytic activity mainly belong to three of these loops, named A, B, and C according to their position in the primary amino acid sequence and all localized at the C-terminal end of the β-barrel. Unlike the rest of the structure, these loops are very flexible and their conformation in a binary complex (enzyme/cofactor) differs from their conformation in a ternary one (enzyme/cofactor/substrate). Their flexibility has functional relevance, since it enables the enzymes to accommodate substrates of varying shapes and sizes and to control many molecular events such as catalytic action. More specifically, members of the aldo-keto reductase (AKR) superfamily2., 3., 4., 5. take advantage of the mobility of some loop motifs to discriminate between many structurally related substrates6., 7., 8., 9., 10., 11. and to coordinate their binding (or release) with that of the cofactor NADP(H).10., 12., 13., 14., 15.
Enzymatic reactions catalyzed by the AKR enzymes follow an ordered bi–bi mechanism in which the cofactor binds first and leaves last.16 Following cofactor binding, the steroid is bound and transformed through a “push–pull” mechanism in which Tyr55 (in the rat liver 3α–HSD), a residue involved in a hydrogen-bond network with residues Lys84 and His117, acts as a proton donor/acceptor.17., 18. Conformational changes undergone by loop B have been found to be a key element in the binding of the cofactor and in its release at the end of the reaction.15., 19. Moreover, recent studies on the structure of the human 20α–HSD (h20α–HSD; AKR1C1) have permitted the identification of a loop B residue (His222), the side-chain of which might mediate the transition between the steroid transformation and cofactor release.18
Human and rabbit 20α–HSDs are highly homologous (80% of amino acid identity) and yet their activity differs. Indeed, h20α–HSD shows a marked preference for the 20α-inactivation of progesterone (Prog) but exerts negligible 3α–HSD and 17β–HSD activities.18 In contrast, the much less specific rabbit 20α–HSD (rb20α–HSD; AKR1C5; 1.1.1.149) binds with similar affinities many steroids presenting different structures.20 Indeed, this enzyme possesses high and comparable catalytic efficiency for the 3α-, 17β-, and 20α-reduction of 5α-dihydrotestosterone (DHT), 4-androstenedione (4-dione) (C19-steroids), and Prog (C21-steroid), respectively (Figure 1).20
To understand the differences between these two 20α–HSDs in their enzyme–substrate interactions and their functionality, crystallographic studies were initiated on human and rabbit enzymes in complex with a few steroid substrates. We have thus determined the crystallographic structure of h20α–HSD in complex with its cofactor and 20α-OHProg, the product of the 20α-reduction of Prog.18 Here, we report the determination of the rb20α–HSD structure in a binary complex with NADPH at a 1.32 Å resolution and in a ternary complex with NADP+ and testosterone (Testo) at a 2.08 Å resolution. Comparison of these two rb20α–HSD structures has revealed significant movements of loop B, which could be crucial for the stabilization of the cofactor and steroid. Indeed, in the binary complex structure, residues of loop B are oriented toward the steroid-binding cavity, likely to protect the hydrophobic character of this site. However, in the presence of the steroid these residues are found at the enzyme surface after a relaxation movement of the loop. Analysis of the geometry of the steroid-binding site of rb20α–HSD in a ternary complex with Testo, a C19-steroid, has allowed us to understand this enzyme ability to bind and transform, with similar efficiency, C18-, C19- and C21-steroids.
Section snippets
Kinetic characterization
Steroid reductases member of the AKR superfamily are known to be involved in the control of active steroid levels in many peripheral tissues and, in doing so, could act as pre-receptor regulators. Whereas human type 3 3α–HSD (AKR1C2; h3α–HSD3) and 20α–HSD show a marked preference for the reduction of ketone groups situated at positions C3 and C2021., 22. of the steroid nucleus, respectively, rb20α–HSD shows an equivalent catalytic efficiency when the ketone group to be reduced is at positions
Conclusion
We have identified three structural elements that could be involved in the reduced selectivity of rb20α–HSD toward its steroid substrates namely: (1) the capacity of loop B to undergo a relaxation movement upon the binding of the steroid substrate, a movement which could be dictated by the nature of the residue at position 230; (2) the nature of the residues found in the C-terminal part of the protein, especially the size of the side-chain of residues at position 306 and 308; and (3) the
Material
Cofactors (NADPH and NADP+) and chemical products were purchased from Sigma-Aldrich Canada. Unlabeled steroids and 14C-labeled steroids were purchased from Steraloids and PerkinElmer, respectively.
Site-directed mutagenesis
Phe54Leu, Phe54Val, Trp227Tyr, Gln230Pro and Val306Phe mutations were created using QuickChange Site-Directed Mutagenesis Kit (Stratagene) with the forward primers listed in Table 5. To confirm the presence of the desired mutation and to ascertain that no other mutation had occurred, the complete
Acknowledgements
This work was supported by the Medical Research Council (MRC) of Canada and Endorecherche Inc. J.-F.C. is the recipient of a doctoral scholarship provided by the Laval University Foundation and Hydro-Québec. P.L. is the recipient of a Post-doctoral fellowship from the Québec Ministry of Education (Bourse d'excellence du Ministère de l'Éducation du Québec). The authors thank Dr D. Poirier for his chemical insight, Dr J. McCarthy for her help using ID14-2 ESRF beam line, Dr V. Nahoum for her help
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Present addresses: J.-F. Couture, Biological chemistry, University of Michigan, 5416 Medical Science Building 1, 1301 Catherine Street, 48109-0606 Ann Arbor, MI, USA; P. Legrand, EMBL Grenoble, 6 rue Jules Horowitz F-38042, Grenoble, Cedex 9, France.