Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand-Binding Domain Revealed by a Combination Andlysis of MD Simulations and SAXS Experiments

Vitamin D receptor (VDR) is a member of the nuclear receptor (NR) family and regulates the expression of genes related to calcium homeostasis , immunomodulation, and cell differentiation and proliferation. The VDR and other NRs are involved in human diseases, therefore, understanding of their regulation by ligand binding is a pharmaceutical demand for structure-based drug design. According to X-ray crystallographic analysis of NRs and other experimental results, the ligand-binding domain (LBD) undergoes conformational change upon ligand binding, and the local conformational change around helix 12 is key to regulating agonism/antagonism.Many crystal structures of agonist/antagonist-binding VDR-LBD have been solved so far. However, all the crystal structures are almost identical, regardless of agonist/antagonist binding, and those crystal structures are considered as the agonist form. Because the conformation of helix 12 is key, the crystal structures do not provide structural insight into the mechanism of antagonist activity. In addition, no crystal structures of apo form has been reported, and the exact conformation of apo form remains unknown.To reveal the apo and antagonist-binding forms of VDR-LBD, we analyze them by a combination approach of small-angle X-ray scattering (SAXS) and molecular dynamics (MD). We obtained SAXS profiles of both forms, however, the profiles were different from the theoretical profiles calculated from crystal structures. To clarify the solution structures at atomic resolution, we conducted MD and collected each structural ensemble. Comparing the SAXS profiles and the MD results, we report a reliable structure for each form. In both forms, helix 12 is partially unraveled, and does not adopt the active form, preventing co-activator binding for transactivation. In apo form, helix 11 bends outward by a kink-centered hinge-bending motion, and the motion creates wide entrance of the ligand-binding pocket. In antagonist-binding form, loop 11-12 remarkably flexible compared to that in apo form.