Ligand-Specific Structural Changes in the Vitamin D Receptor in Solution

Abstract

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogs, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogs cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a potent agonist analog, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D3 [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of co-activators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and 12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of co-activators.

Evolutionary and Functional Diversification of the Vitamin D Receptor-Lithocholic Acid Partnership

“…These results suggest that basal species may lack the ability to form transcriptionally active conformations with LCA. This is likely due to an inability of LCA to re-localize helices H2, H3, H11 and H12 which induces a structural transition that triggers a mousetrap-like mechanism and stabilizes ligand binding and co-regulator recruitment [45]. Future studies to determine the crystal structures of basal VDRs or zebrafish VDRβ complexed with LCA may be very informative with regards to revealing species-specific differences in VDR conformations associated the observed functional differences.…”

Comparative Metabolomics Reveals Endogenous Ligands of DAF-12, a Nuclear Hormone Receptor, Regulating C. elegans Development and Lifespan

“…Screening of synthetic ligands of the vitamin D receptor and other mammalian NHRs has demonstrated that even small changes in ligand structures can strongly affect gene transcription (Brown and Slatopolsky, 2008; Singarapu et al, 2011; Wollam and Antebi, 2011). Yet there are few approaches to comprehensively identify the endogenous NHR ligands from complex animal metabolomes, whose chemical annotations remain largely incomplete.…”

“…Precise knowledge of ligand structures and biosynthetic pathways is essential for understanding NHR function (Mangelsdorf et al, 1995; Wollam and Antebi, 2011), because even small differences in ligand structures may result in dramatic changes of transcriptional activity and specificity (Brown and Slatopolsky, 2008; Singarapu et al, 2011). However, the endogenous ligands of many NHRs remain poorly characterized, in part because ligands constitute very minor components of highly complex animal metabolomes (Schupp and Lazar, 2010).…”

Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies

“…A 4-fold molar excess of the 13-residue peptide from DRIP205, which contains the LxxLL motif mimicking the co-activator was added to the final NMR sample [43] prior to data collection. To better estimate level of perdeuteration, TROSY (transverse relaxation-optimized spectroscopy) based 15 N resolved 3D- 1 H, 1 H NOESY spectra were collected.…”