Structural and functional heterogeneity among the zinc fingers of human MRE-binding transcription factor-1
Structural and functional heterogeneity among the zinc fingers of human MRE-binding transcription factor-1
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DOI：
10.1021/bi980843r
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发表时间：
1998-08-11
期刊：
影响因子：
2.9
通讯作者：
Giedroc, DP
中科院分区：
文献类型：
--
作者：
Chen, XH;Agarwal, A;Giedroc, DP
MRE 结合转录因子-1 (MTF-1) 在与 MT 启动子中的一个或多个串联金属反应元件（MRE；5'-ctnTGCRCnCgGCCc）结合后，激活小鼠和人类细胞中金属硫蛋白 (MT) 基因的表达。 MTF-1 包含六个 Cys(2)-His(2) 锌指序列。先前的工作表明，锌指结构域本身可能在锌激活的 MT 表达中充当锌传感器。为了从分子角度深入了解 MTF-1 功能，使用非变性条件纯化了仅包含锌指结构域的 MTF-1 重组片段（表示为 MTF-zf），并对其锌结合特性、二级结构和 DNA 结合活性进行了表征。 MTF-zf 的不同制剂，在厌氧透析后定量 Zn(II) 和还原半胱氨酸（通过 DTNB 反应性）含量，揭示了 Zn(II) 和 11-13 还原硫醇盐（预期为 12）的 Zn(II)/MTF-zf 化学计量范围为 3.3 至 5.5 g。远紫外圆二色光谱揭示了所有制剂中无法区分的二级结构含量，即足以折叠 MTF-zf 的六个锌指中的三个。从 MTF-zf 中去除额外的锌会产生不溶性脱辅基蛋白。通过香豆素荧光各向异性的变化监测，Zn-5.5 MTF-zf 和香豆素标记的含有 MREd 的寡核苷酸之间形成复合物，得出 K-app = 3.8 (+/-0.5) x 10(8) M-1（pH 7.0，0.20 M NaCl，25 摄氏度）。对 K-app 的盐类型和浓度依赖性的研究表明，复合物形成时阳离子和阴离子的释放具有显着贡献。 Zn-5.5 MTF-zf 表现出与 MREd 形成复合物的大负热容，并且可以区分在 TGCRC 核心或 MREd 富含 C 侧沉积有突变的 DNA 双链体。 Zn-5.5 MTF-zf 的空气氧化导致 12 个 Cys 硫醇盐中的 6 个可逆转化为 3 个二硫键；正如预期的那样，这对 MTF-zf 的二级结构没有影响，但导致 K-app 减少约 30 倍，接近 1.2 x 10(7) M-1。相反，完全还原的 Zn-3.5 MTF-zf 与 MREd 的结合亲和力和 [NaCl] 依赖性与 Zn-5.5 MTF-zf 的亲和力和 [NaCl] 依赖性基本上无法区分。 MTF-zf 中的锌指在物理和功能上是不等价的，锌指的一个子集（大约 3-4 个）在折叠和高亲和力 MREd 结合中发挥结构作用，而一个或多个附加指具有可能与金属调节作用一致的特性。
MRE-binding transcription factor-1 (MTF-1) activates the expression of metallothionein (MT) genes in mouse and human cells upon binding to one or more tandem metal-response elements (MREs; 5'-ctnTGCRCnCgGCCc) in the MT promoter. MTF-1 contains six Cys(2)-His(2) zinc finger sequences. Previous work suggests that the zinc finger domain itself may function as a zinc sensor in zinc-activated expression of MTs. To obtain molecular insight into MTF-1 function, a recombinant fragment of MTF-1 containing only the zinc finger domain (denoted MTF-zf) has been purified using nondenaturing conditions and characterized with respect to zinc-binding properties, secondary structure, and DNA-binding activity. Different preparations of MTF-zf, following an anaerobic dialysis to quantify Zn(II) and reduced cysteine (by DTNB reactivity) content, reveal Zn(II)/MTF-zf stoichiometries ranging from 3.3 to 5.5 g at Zn(II) and 11-13 reduced thiolates (12 expected). Far-UV CD spectra reveal indistinguishable secondary structural content in all preparations, i.e., enough to fold just three of six zinc fingers of MTF-zf. Removal of additional zinc from MTF-zf gives rise to an insoluble apoprotein. Complex formation between a Zn-5.5 MTF-zf and a coumarin-labeled MREd-containing oligonucleotide as monitored by changes in the anisotropy of the coumarin fluorescence gives a K-app = 3.8 (+/-0.5) x 10(8) M-1 (pH 7.0, 0.20 M NaCl, 25 degrees C). Investigation of the salt type and concentration dependence of K-app suggests significant contributions from both cation and anion release upon complex formation. Zn-5.5 MTF-zf exhibits a large negative heat capacity of complex formation with MREd and can discriminate among DNA duplexes which have mutations deposited on either the TGCRC core or the C-rich side of the MREd. Air oxidation of Zn-5.5 MTF-zf results in the reversible conversion of 6 of the 12 Cys thiolates to 3 disulfide bonds; as expected, this has no effect on the secondary structure of MTF-zf, but results in approximate to 30-fold reduction in K-app to approximate to 1.2 x 10(7) M-1. In contrast, fully reduced Zn-3.5 MTF-zf binds to the MREd with an affinity and [NaCl] dependence largely indistinguishable from those of Zn-5.5 MTF-zf. The zinc fingers in MTF-zf are physically and functionally inequivalent, A subset (approximate to 3-4) of zinc fingers plays a structural role in folding and high-affinity MREd binding, while one or more additional fingers have properties potentially consistent with a metalloregulatory role.