ZINC-BINDING PROPERTIES OF THE DNA-BINDING - DOMAIN OF THE 1,25-DIHYDROXYVITAMIN D-3 RECEPTOR

TO assess the zinc binding stoichiometry and the structural changes in duced 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 ape-protein and metal-liganded protein by mass spec trometry, and circular dichroism and nuclear magnetic resonance spectr oscopy. Following final preparation with a zinc-free buffer, the VDR D BD bound 2 mel of zinc/mol of protein as measured by inductively coupl ed plasma-mass spectrometry and electrospray ionization-mass spectrome try. When protein preparation was carried out in a zinc containing buf fer and zinc content of the protein was assesed by the same methods, V DR DBD bound 4 mel of zinc/mol of protein. Analysis of the protein usi ng circular dichroism spectroscopy demonstrated that the EDTA-treated protein increased in alpha-helical content from 16 to 27% on the addit ion of zinc. Equilibrium ultracentrifugal analyses of the VDR DBD indi cated that the protein was present in solution as a monomer, Gel mobil ity shift analyses of the VDR DBD with several vitamin D response elem ents (VDREs) in the absence of accessory proteins such as retinoic aci d receptor, showed that VDR DBD was able to form a protein/VDRE DNA st ructural complex. In the presence of zinc, proton NMR NOESY spectra sh owed that the protein possessed elements of secondary structure, The a ddition of VDRE DNA, but not random DNA, caused changes in the proton NMR spectra of VDRE DNA indicating specific interaction between protei n and DNA groups. We conclude that the DBD of the VDR binds zinc and D NA and undergoes conformational changes on binding to the metal and DN A.