MRE-binding transcription factor-1: Weak zinc-binding finger domains 5 and 6 modulate the structure, affinity, and specificity of the metal-response element complex
MRE-binding transcription factor-1: Weak zinc-binding finger domains 5 and 6 modulate the structure, affinity, and specificity of the metal-response element complex
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DOI：
10.1021/bi9913000
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发表时间：
1999-09-28
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;Chu, MH;Giedroc, DP
MRE 结合转录因子-1 (MTF-1) 包含六个 Cys(2)-His(2) 锌指序列，有人提出锌指结构域本身可能在锌激活的金属硫蛋白 (MT) 表达中充当锌传感器。先前的工作表明，MTF-zf 中的锌指的一个子集（大约 3-4 个）在折叠和高亲和力金属响应元件（MREd）结合中发挥结构作用，而一个或多个其他指具有与金属调节作用一致的特性（在缺乏 DNA 的情况下锌结合亲和力较弱）。我们在此表明锌指 5 和 6 对应于 MTF-zf 中的弱锌结合指。 Zn-6-MTF-zf:MREd 复合物的有限胰蛋白酶分解产生与 MTF-zf 的氨基酸 137-260 或 N 端锌指 1-4 相对应的高度蛋白酶抗性核心片段。对 MTF-zf 断指 (His --> Asn) 和缺失指突变体集合的表征表明，删除锌指 5 和 6 以产生 MTF-zf14 会减弱 MREd 结合亲和力（大约 20 倍），而删除指 4-6 (MTF-zf13) 会导致结合亲和力进一步降低 20 倍，且特异性几乎完全丧失。圆二色性研究表明，MTF-zf 与 MREd 的结合会导致 MREd 结构发生显着变化，从 B 型变为具有 A 样特征的双螺旋构象。与 MTF-2f14、H279N (Delta zf5) MTF-zf 和 MTF-zf13 形成化学计量复合物会诱导相对较少的 A 样结构。稳态荧光共振能量转移 (FRET) 光谱已用于全局定义 MREd 上多指 MTF-zf 的方向。这些实验表明，指1-4定向于MREd的高度保守的TGCRCnC侧，而指5-6结合在gGCCc序列处或附近。这些发现与模型一致，其中 MTF-zf 中的 N 端锌指需要高亲和力和特异性结合到共有 TGCRCnC 核心，其方式受到弱锌结合 C 端锌指的结构和变构调节。
MRE-binding transcription factor-1 (MTF-1) contains six Cys(2)-His(2) zinc finger sequences, and it has been suggested that the zinc finger domain itself may function as a zinc sensor in zinc-activated expression of metallothioneins (MTs). Previous work has shown that a subset (approximate to 3-4) of the zinc fingers in MTF-zf play a structural role in folding and high-affinity metal-response element (MREd) binding, while one or more other fingers have properties consistent with a metalloregulatory role (weak zinc binding affinity in the absence of DNA). We show here that zinc fingers 5 and 6 correspond to the weak zinc-binding fingers in MTF-zf. Limited trypsinolysis of a Zn-6-MTF-zf:MREd complex gives rise to a highly protease-resistant core fragment corresponding to amino acids 137-260 or N-terminal zinc fingers 1-4 of MTF-zf. Characterization of a collection of broken-finger (His --> Asn) and missing-finger mutants of MTF-zf reveals that deletion of zinc fingers 5 and 6 to create MTF-zf14 attenuates MREd binding affinity (approximate to 20-fold), while deletion of fingers 4-6 (MTF-zf13) results in a further 20-fold reduction of binding affinity with a nearly complete loss of specificity. Circular dichroism studies reveal that the binding of MTF-zf to the MREd induces a dramatic alteration of the structure of the MREd from a B-form to a double-helical conformation with A-like features. Formation of stoichiometric complexes with MTF-2f14, H279N (Delta zf5) MTF-zf, and MTF-zf13 induces comparatively less A-like structure. Steady-state fluorescence resonance energy transfer (FRET) spectroscopy has been used to globally define the orientation of the multifinger MTF-zf on the MREd. These experiments suggest that fingers 1-4 are oriented on the highly conserved TGCRCnC side of the MREd with fingers 5-6 bound at or near the gGCCc sequence. These findings are consistent with a model in which the N-terminal zinc fingers in MTF-zf are required for high affinity and specific binding to the consensus TGCRCnC core in a way which is subjected to structural and allosteric modulation by the weak zinc-binding C-terminal zinc fingers.