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 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). 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)(2)D(3) 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)(2)D(3). Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)(2)D(3)-1-BE), Cys288 (beta-hairpin region: 1,25(OH)(2)D(3)-3-BE,) and Tyr295 (helix-6: 1,25(OH)(2)D(3)-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the beta-hairpin region (modified by 1,25(OH)(2)D(3)-3-BE) is most important for growth inhibition by 1,25(OH)(2)D(3), while helices 3 and 6 are less important for such activity.
Kaya
Taner
T
Boston University, Boston, MA 02215, USA.
Swamy
Narasimha
N
Persons
Kelly S
KS
Ray
Swapna
S
Mohr
Scott C
SC
Ray
Rahul
R
D016475:3T3 Cells; D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D000477:Alkylating Agents / Q000138:chemical synthesis / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D002460:Cell Line; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
C540872:1,25-dihydroxyvitamin D3-1-(2)-bromoacetate; C540873:1,25-dihydroxyvitamin D3-11-hydroxy-(2)-bromoacetate; C104313:1,25-dihydroxyvitamin D3-3-bromoacetate; D000345:Affinity Labels; D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D008024:Ligands; D018167:Receptors, Calcitriol; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D002117:Calcitriol