Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor.
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 overexpressed 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., & Yamamoto, K. R. (1988) Nature 334, 543]. Zn(II) [or Cd(II) substitution] has been shown to be essential for specific DNA binding. 113Cd NMR 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) identifies 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Pan
T
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Freedman
L P
LP
Coleman
J E
JE
D000595:Amino Acid Sequence; D001665:Binding Sites; D002106:Cadmium Radioisotopes; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism; D016335:Zinc Fingers
D002106:Cadmium Radioisotopes; D011965:Receptors, Glucocorticoid; D006859:Hydrogen; D004247:DNA