Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor

Publisher Website
Google Scholar
Abstract
The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entire DNA binding domain (residues 440-525), has been overexposed and purified from Escherichia coli previously. This fragment has been shown to contain 2.3 .+-. 0.2 mol of Zn(II) per mole of protein [Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., and Yamamoto, K. R. (1988) Nature 334, 543]. Zn(II) [or Cd(II) substitution] has been shown to be essential for specific DNA binding. 113Cd NRM of a cloned construct containing the minimal DNA binding domain of 86 AA residues [denoted GR (440-525)] with 113Cd(II) substituted for Zn(II) identified 2 Cd(II) binding sites by the presence of 2 113Cd NMR signals each of which integrates to 1 113Cd nucleus. The chemical shifts of these two sites, 704 and 710 ppm, suggest that each 113Cd(II) is coordinated to four isolated -S- ligands. Shared -S- ligands connecting the two 113Cd(II) ions do not appear to be present, since their T1s differ by 10-fold, 0.2 and 2.0 s, respectively. Addition of a third 113Cd(II) or Zn(II) to 113Cd2GR(440-525) results in occupancy of a third site, which introduces exchange modulation of the two original 113Cd NMR signals causing them to disappear. Addition of EDTA to the protein restores the original two signals. 1H-113Cd heteronuclear multiple quantum spectroscopy of GR(440-525) shows that the major protons coupled to 113Cd are a group of .beta.-protons assignable to Cys residues. A small variable signal corresponding to a .epsilon.-CH3 of Met coupled to 113Cd suggests that the thioether of a Met may be a ligand to the third 113Cd(II) ion. Binding of the third 113Cd(II) causes significant increases of the Cd-S charge transfer absorption bands. We propose that GR(440-525) can form three Zn(II) binding sites involving a combination of the nine Cys and one Met residues as ligands, all of which are highly conserved among the superfamily of steroid and thyroid hormone receptors. Both circular dichroism and 1H NMR show the folding of GR(440-525) to be dependent on the presence of Zn(II) or Cd(II). Removal of the metal ions causes GR(440-525) to completely unfold from its native structure. Both Zn(II) and Cd(II)GR(440-525) have very similar 1H NMR spectra, suggesting almost identical structures of the two metal derivatives of the DNA binding domain of GR.