Zinc Binding Properties of the DNA Binding Domain of the 1,25-Dihydroxyvitamin D3 Receptor
To assess the zinc binding stoichiometry and the structural changes induced upon the binding of zinc to the human vitamin D receptor (VDR), we expressed the DNA binding domain (DBD) of the human VDR in bacteria as a soluble glutathione-S-transferase fusion protein at 20 degrees C, and examined the apo-protein and metal-liganded protein by mass spectrometry, and circular dichroism and nuclear magnetic resonance spectroscopy. Following final preparation with a zinc-free buffer, the VDR DBD bound 2 mol of zinc/mol of protein as measured by inductively coupled plasma-mass spectrometry and electrospray ionization-mass spectrometry. When protein preparation was carried out in a zinc containing buffer and zinc content of the protein was assesed by the same methods, VDR DBD bound 4 mol of zinc/mol of protein. Analysis of the protein using circular dichroism spectroscopy demonstrated that the EDTA-treated protein increased in alpha-helical content from 16 to 27% on the addition of zinc. Equilibrium ultracentrifugal analyses of the VDR DBD indicated that the protein was present in solution as a monomer. Gel mobility shift analyses of the VDR DBD with several vitamin D response elements (VDREs) in the absence of accessory proteins such as retinoic acid receptor, showed that VDR DBD was able to form a protein/VDRE DNA structural complex. In the presence of zinc, proton NMR NOESY spectra showed that the protein possessed elements of secondary structure. The addition of VDRE DNA, but not random DNA, caused changes in the proton NMR spectra of VDRE DNA indicating specific interaction between protein and DNA groups. We conclude that the DBD of the VDR binds zinc and DNA and undergoes conformational changes on binding to the metal and DNA.
Amino acid sequence, base sequence, binding sites, circular dichroism, cloning, molecular, DNA, DNA-binding proteins, electrophoresis, polyacrylamide gel, humans, magnetic resonance spectroscopy, mass spectrometry, oligodeoxyribonucleotides, peptide fragments, protein conformation, receptors, calcitriol, recombinant fusion proteins, ultracentrifugation, zinc
Craig, Theodore A.; Veenstra, Timothy D.; Naylor, Stephen; Tomlinson, Andy J.; Johnson, Kenneth L.; Macura, Slobodan; Juranić, Nenad; and Kumar, Rajiv, "Zinc Binding Properties of the DNA Binding Domain of the 1,25-Dihydroxyvitamin D3 Receptor" (1997). Pharmaceutical Sciences Faculty Publications. 544.