Ratiometric pulsed Alkylation/Mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-Binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability
Ratiometric pulsed Alkylation/Mass spectrometry of the cysteine pairs in individual zinc fingers of MRE-Binding transcription factor-1 (MTF-1) as a probe of zinc chelate stability
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DOI：
10.1021/bi0112208
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发表时间：
2001-12-18
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：
Apuy, JL;Chen, XH;Giedroc, DP
金属反应元件结合转录因子-1(MTF-1)是一种锌调控的哺乳动物细胞金属硫蛋白(MT)基因转录激活因子。MTF-1的MRE结合域(MTF-ZF)有六个典型的Cys(2)-His(2)锌指结构域，根据它们与锌的表观亲和力和它们在MRE结合中的特定作用来区分。本文中，锌配体半胱氨酸硫酸酯对与巯基特异性烷基化试剂d(5)-N-乙基马来酰亚胺(d(5)-NEM)的脉冲烷基化反应被用来作为锌指配位络合物在锌-6MTF-ZF中相对稳定性的残基特异性探针。用过量的H-5-N-乙基马来酰亚胺(HS-NEM)完全衍生MTF-ZF伴随着完全的蛋白质分解，然后进行MALDI-TOF质谱分析，可以作为d(5)-NEM脉冲时间的函数来定量每个锌指结构域对应的d(5)、d(5)-、d(5)、H-5-和H-5，H-5-NEM衍生多肽的摩尔分数。本实验建立了MTF-ZF中半胱氨酸硫酸盐反应性的等级为F5>；F6远大于F1>；F2近似于F3近似于F4。在相同的溶液条件下，F_1硫代化合物的表观二级反应速率与测定的Sp1，Zn-3SP1-ZF的DNA结合域的表观二级反应速率相当。当与含有MRED的寡核苷酸结合时，MTF-ZF中所有半胱氨酸残基的反应性显著降低。与缺少N-末端F1锌指的MTF-ZF结构域Zn-5 MTF-zf26进行的相同实验表明，MTF-zf26与MRED结合非常弱，其特征是非相邻F4硫酸盐的反应活性显著增加。这些发现在现有的MTF-1金属调节模型的背景下进行了讨论。
Metal-response element (MRE)-binding transcription factor-1 (MTF-1) is a zinc-regulated transcriptional activator of metallothionein (MT) genes in mammalian cells. The MRE-binding domain of MTF-1 (MTF-zf) has six canonical Cys(2)-His(2) zinc finger domains that are distinguished on the basis of their apparent affinities for zinc and their specific roles in MRE-binding. In this paper, pulsed alkylation of the zinc-liganding cysteine thiolate pairs with the sulfhydryl-specific alkylating reagent d(5)-N-ethylmaleimide (d(5)-NEM) is used as a residue-specific probe of the relative stabilities of the individual zinc finger coordination complexes in Zn-6 MTF-zf. A chase with excess H-5-N-ethylmaleimide (HS-NEM) to fully derivatize MTF-zf concomitant with complete proteolysis, followed by MALDI-TOF mass spectrometry allows quantitation of the mole fraction of d(5),d(5)-, d(5),H-5-, and H-5,H-5-NEM derivatized peptides corresponding to each individual zinc finger domain as a function of d(5)-NEM pulse time. This experiment establishes the hierarchy of cysteine thiolate reactivity in MTF-zf as F5 > F6 much greater than F1 > F2 approximate to F3 approximate to F4. The apparent second-order rate of reaction of Fl thiolates is comparable to that determined for the DNA binding domain of Sp1, Zn-3 SP1-zf, under identical solution conditions. The reactivities of all Cys residues in MTF-zf are significantly reduced when bound to an MREd-containing oligonucleotide. An identical experiment carried out with Zn-5 MTF-zf26, an MTF-zf domain lacking the N-terminal F1 zinc finger, reveals that MTF-zf26 binds to the MREd very weakly, and is characterized by strongly increased reactivity of nonadjacent F4 thiolates. These findings are discussed in the context of existing models for metalloregulation by MTF-1.