Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor.
Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor.
复制标题
哺乳动物糖皮质激素受体 DNA 结合域的镉 113 NMR 研究。
DOI：
10.1021/bi00491a016
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Coleman,JE
中科院分区：
文献类型：
--
作者：
Pan,T;Freedman,LP;Coleman,JE
耶鲁大学分子生物学和生物化学系，纽黑文，康涅狄格州06510，和加州大学生物化学和生物物理学系，弗朗西斯科，加州94143，1990年3月30日接收;修订版手册，1990年6月26日接收摘要：哺乳动物糖皮质激素受体（GR）的DNA结合结构域含有9个高度保守的半胱氨酸残基，类固醇和甲状腺激素受体超家族共有的保守性。由GR残基407-556组成的片段[长度为150个氨基酸（AA）]，其中含有整个DNA结合结构域（残基440-525），先前已从大肠杆菌中过表达并纯化。该片段已被证明每摩尔蛋白质含有2.3±0.2摩尔Zn（II）[Freedman，L. P.，Luisi，BF，Korszun，Z. R.，巴萨瓦帕河Sigler，P. B.，& Yamamoto，K. R.（1988）Nature 334，543]。Zn（II）[或Cd（II）取代]已被证明是特异性DNA结合所必需的。用~（113）Cd（Ⅱ）取代Zn（Ⅱ）的86个氨基酸残基的最小DNA结合结构域[记为GR（440-525）]的克隆构建体的~（113）Cd NMR通过存在2个~（113）Cd NMR信号（每个信号整合到~（113）Cd核）鉴定出2个Cd（Ⅱ）结合位点。704和710 ppm的化学位移表明，每个~（113）Cd（II）与四个孤立的-S ~-配体配位。连接两个U3 Cd（II）离子的共享-S ~配体似乎不存在，因为它们的π s分别相差10倍、0.2和2.0 s。将第三个U3 Cd（II）或Zn（II）添加到U3 Cd 2GR（440-525）导致第三个位点的占据，这引入了两个原始113 Cd NMR信号的交换调制，导致它们消失。向蛋白质中添加EDTA恢复了原始的两个信号。GR（440-525）的~（113）H ~+-Cd杂原子多重量子谱表明，与~（113）Cd偶联的主要质子是一组可归属于Cys残基的O-质子。一个小的可变信号对应于一个电子-CH 3的Met耦合，13镉表明，甲硫氨酸的硫醚可能是一个配体的第三个113镉（II）离子。第三个~（1 I）3Cd（II）的结合导致Cd-S电荷转移吸收带显著增加。我们认为GR（440-525）可以形成三个Zn（II）结合位点，包括九个Cys和一个Met残基作为配体，它们在类固醇和甲状腺激素受体超家族中高度保守。圆二色性和NMR都显示GR（440-525）的折叠依赖于Zn（II）或Cd（II）的存在。金属离子的去除导致GR（440-525）从其天然结构完全展开。Zn（II）和Cd（II）GR（440-525）的NMR谱非常相似，表明GR的DNA结合结构域的两种金属衍生物的结构几乎相同。
Department of Molecular Biophysicsand Biochemistry, Yale University, New Haven, Connecticut 06510, and Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143 Received March 30, 1990; Revised Manuscript Received June 26, 1990 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 overexpressed and purified from Escherichia coli previously. This fragment has been shownto contain 2.3±0.2 mol of Zn (II) per mole of protein [Freedman, L. P., Luisi, BF, 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 specificDNA binding. 113Cd NMR of a cloned construct containing the minimal DNA binding domain of 86 AA residues [denoted GR (440-525)] with u3Cd (II) substituted for Zn (II) identifies 2 Cd (II) binding sites by the presence of 2 113Cd NMR signals each of which integratesto 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 U3Cd (II) ions do not appear to be present, since their 7js differ by 10-fold, 0.2 and 2.0 s, respectively. Addition of a third U3Cd (II) or Zn (II) to U3Cd2GR (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.'H-'^ Cd heteronuclear multiple quantum spectroscopy of GR (440-525) shows that the major protons coupledto 113Cd are a group of 0-protons assignable to Cys residues. A small variable signal corresponding to a e-CH3 of Met coupled to, 13Cd suggests that the thioether of a Met may be a ligand to the third 113Cd (II) ion. Binding of the third 1I3Cd (II) causes significant increase of the Cd-S charge transfer absorption bands. We propose thatGR (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 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 NMR spectra, suggesting almost identical structuresfor the two metal derivatives of the DNA binding domain of GR.