Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: Structural and biochemical implications

Structure–functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter ‘surface structure’ of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)2D3 containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)2D3. Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)2D3-1-BE), Cys288 (β-hairpin region: 1,25(OH)2D3-3-BE,) and Tyr295 (helix-6: 1,25(OH)2D3-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the β-hairpin region (modified by 1,25(OH)2D3-3-BE) is most important for growth inhibition by 1,25(OH)2D3, while helices 3 and 6 are less important for such activity.