Background: Bisubstrate enzymes, such as 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1), exist in solution as an ensemble of conformations. 17b-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. Methodology/Principal Findings: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17b-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. Conclusions/Significance: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/complex approach revealed an essential role played by the backbone and side chain motions, especially of the bFaG9-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17b-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway
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