Presence of a copper(I)-thiolate regulatory domain in the copper-activated transcription factor Amt1
Presence of a copper(I)-thiolate regulatory domain in the copper-activated transcription factor Amt1
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DOI：
10.1021/bi961642v
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发表时间：
1996-11-19
期刊：
影响因子：
2.9
通讯作者：
Winge, DR
中科院分区：
文献类型：
--
作者：
Graden, JA;Posewitz, MC;Winge, DR
来自光滑念珠菌的 Amt1 转录因子通过四铜硫醇簇的形成而被激活。重组 Amt1（残基 1-110）被分离为 Cu,ZnAmt1 复合物。先前的作图研究[Farrell 等人，(1996) Biochemistry 35, 1571-1580]揭示了Zn(II)位点被由残基1-40组成的独立的N端结构域包围。绘图研究的一项预测是四铜簇被残基 41-110 包围。由残基 37-110 组成的截短的 Amt1 肽被表达并分离为与 4 摩尔当量结合的 Cu(I) 的 CuAmt1 复合物。 The bound Cu(I) ions in the truncated Amt1 complex were spectroscopically similar to Cu(I) ions bound in the 110-mer Amt1 molecule in the energies and intensities of the ultraviolet S --> Cu charge transfer transitions and luminescence.截短的 CuAmt1 配合物的铜 K 边扩展 X 射线吸收精细结构光谱 (EXAFS) 显示与 Cu,ZnAmt1 配合物中相同的 2.26 埃平均 Cu-S 键距。 Cu,ZnAmt1 中的聚硫醇铜簇的一个诊断特征是由 Cu K 边缘 EXAFS 确定的短 2.7 埃 Cu-Cu 距离。截短的 CuAmt1 复合物具有同样短的 2.7 埃 Cu-Cu 距离。截短的 CuAmt1 复合物以高亲和力特异性结合 DNA，与残基 41-110 一致，残基 41-110 是由四铜簇稳定的独立结构域。因此，Amt1 由三个独立且连续的结构域组成：N 端 Zn 模块（残基 1-40）、相邻的 Cu 调节结构域（残基 41-110）和 C 端转录激活结构域。 Amt1 的 Cu(I) 激活似乎包括 70 个残基的 Cu 调节结构域从非活性构象异构体转化为包含四铜簇的结构。
The Amt1 transcription factor from Candida glabrata is activated by the formation of a tetracopper-thiolate cluster. Recombinant Amt1 (residues 1-110) is isolated as a Cu,ZnAmt1 complex. Previous mapping studies [Farrell et al, (1996) Biochemistry 35, 1571-1580] revealed that the Zn(II) site is enfolded by an independent, N-terminal domain consisting of residues 1-40. One prediction from the mapping study is that the tetracopper cluster is enfolded by residues 41-110. A truncated Amt1 peptide consisting of residues 37-110 was expressed and isolated asa CuAmt1 complex with 4 mol equiv of Cu(I) bound. The bound Cu(I) ions in the truncated Amt1 complex were spectroscopically similar to Cu(I) ions bound in the 110-mer Amt1 molecule in the energies and intensities of the ultraviolet S --> Cu charge transfer transitions and luminescence. Copper K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) of the truncated CuAmt1 complex revealed the same 2.26 Angstrom mean Cu-S bond distance as in the Cu,ZnAmt1 complex. A diagnostic feature of the polycopper-thiolate cluster in Cu,ZnAmt1 is the short 2.7 Angstrom Cu-Cu distance determined by Cu K-edge EXAFS. The truncated CuAmt1 complex had the same short 2.7 Angstrom Cu-Cu distance. The truncated CuAmt1 complex bound DNA specifically and with high affinity consistent with residues 41-110 being an independent domain stabilized by the tetracopper cluster. Thus, Amt1 consists of three independent and contiguous domains, an N-terminal Zn module (residues 1-40), an adjacent Cu regulatory domain (residues 41-110), and a C-terminal transcriptional activation domain. Cu(I) activation of Amt1 appears to consist of conversion of the 70-residue Cu regulatory domain from an inactive conformer to a structure containing the tetracopper cluster.