Functional and structural characterization of nuclear vitamin D receptor and its ligand binding domain

Abstract The hormonally active form of vitamin D, 1,25(OH)2D3, is involved in many biological functions throughout the body, such as regulation of calcium and phosphate homeostasis, bone remodeling and controlling cell proliferation and differentiation. Vitamin D receptor (VDR), a member of the nuclear hormone receptor (NHR) super family, mediates those genomic actions of 1,25 (OH)2D3 by actively repressing or activating its target genes. In the present study recombinant human nuclear VDR and its ligand binding domain (LBD) were expressed in Spodoptera frugiperda (Sf9) insect cells and in E.coli. Recombinant proteins were purified and their biochemical and biophysical properties were characterized. Recombinant VDR was shown to bind to the vitamin D response element (VDRE) of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor (RXR)-αΔAB. Full-length VDR and its LBD were demonstrated to bind natural ligand 1,25 (OH)2D3 with high affinity. The binding affinities of several vitamin D analogs were also determined. Ligand binding induced conformational change within the receptor was studied using several methods such as partial proteolytic digestion, small angle neutron scattering (SANS), native gel electrophoresis and circular dichroism (CD) spectroscopy. Results indicate that ligand binding induces conformational change within VDR and different 1,25(OH)2D3 analogs might induce a somewhat different conformation within the receptor. This is seen as an unequal capacity of analogs to stabilize receptor against proteases or heat and as differences in the promotion of receptor homodimerization. Compared to other nuclear hormone receptors, VDR presents a large insertion region at the N-terminal part of the LBD between helices H1 and H3, encoded by an additional exon. In the present study this additional exon was deleted and the properties of mutated LBD were compared to the wild type LBD. Biochemical analyses indicated that the mutant protein exhibits the same ligand binding, dimerization with RXR and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Furthermore, solution studies by small angle X-ray scattering indicated that the insertion region in the VDR locates on the surface of molecule and it is not structurally well ordered