Zinc binding properties of the DNA binding domain of the 1,25- dihydroxyvitamin D3 receptor

Theodore A. Craig, Timothy D. Veenstra, Stephen Naylor, Andy J. Tomlinson, Kenneth L. Johnson, Slobodan I Macura, Nenad Juranić, Rajiv Kumar

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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°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 α-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.

Original languageEnglish (US)
Pages (from-to)10482-10491
Number of pages10
JournalBiochemistry
Volume36
Issue number34
DOIs
StatePublished - Aug 26 1997

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Calcitriol Receptors
Zinc
DNA
Vitamin D Response Element
Proteins
Circular Dichroism
Mass spectrometry
Protons
Mass Spectrometry
Buffers
Metals
Nuclear magnetic resonance
Circular dichroism spectroscopy
Inductively coupled plasma mass spectrometry
Electrospray ionization
Retinoic Acid Receptors
Electrospray Ionization Mass Spectrometry
Accessories
Electrophoretic Mobility Shift Assay
Glutathione Transferase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zinc binding properties of the DNA binding domain of the 1,25- dihydroxyvitamin D3 receptor. / Craig, Theodore A.; Veenstra, Timothy D.; Naylor, Stephen; Tomlinson, Andy J.; Johnson, Kenneth L.; Macura, Slobodan I; Juranić, Nenad; Kumar, Rajiv.

In: Biochemistry, Vol. 36, No. 34, 26.08.1997, p. 10482-10491.

Research output: Contribution to journalArticle

Craig, TA, Veenstra, TD, Naylor, S, Tomlinson, AJ, Johnson, KL, Macura, SI, Juranić, N & Kumar, R 1997, 'Zinc binding properties of the DNA binding domain of the 1,25- dihydroxyvitamin D3 receptor', Biochemistry, vol. 36, no. 34, pp. 10482-10491. https://doi.org/10.1021/bi970561b
Craig TA, Veenstra TD, Naylor S, Tomlinson AJ, Johnson KL, Macura SI et al. Zinc binding properties of the DNA binding domain of the 1,25- dihydroxyvitamin D3 receptor. Biochemistry. 1997 Aug 26;36(34):10482-10491. https://doi.org/10.1021/bi970561b
Craig, Theodore A. ; Veenstra, Timothy D. ; Naylor, Stephen ; Tomlinson, Andy J. ; Johnson, Kenneth L. ; Macura, Slobodan I ; Juranić, Nenad ; Kumar, Rajiv. / Zinc binding properties of the DNA binding domain of the 1,25- dihydroxyvitamin D3 receptor. In: Biochemistry. 1997 ; Vol. 36, No. 34. pp. 10482-10491.
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