Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1)
Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1)
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DOI：
10.1021/bi0358418
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发表时间：
2004-05-11
期刊：
影响因子：
2.9
通讯作者：
Berg, JM
中科院分区：
文献类型：
--
作者：
Guerrerio, AL;Berg, JM
金属反应元件（MRE）结合转录因子-1 （MTF1）是一种6 Cys(2)His(2)含锌指的转录因子，是金属硫蛋白基因基础转录和锌诱导转录所必需的。由6个锌指结构域组成的蛋白质片段的钴（II）和锌（11）的亲和性已经被检测，揭示了钴（II）的表观解离常数为0.5 +/- 0.2 ma，锌（11）的31 +/- 14 pM。两种方法已被用来确定金属离子亲和的个别领域。首先，这六个结构域作为单域肽进行了研究，揭示了钴（II）的解离常数从0.3到1.7 muM不等。结构域分为两组，结构域2、3和4对应的肽具有较高的亲和力（K-d(Co(It)) 0.3-0.5 muM），结构域1、5和6对应的肽具有较低的亲和力（K-d(Co(II)) 1.6-1.7 muM）。其次，我们通过单独将一个金属结合的His残基突变为Cys来检测六个锌指结构域蛋白中每个结构域的亲和力，从而独立监测每个位点的钴（IT）占用情况。在这种情况下，每个结构域的亲和力比单个结构域肽高，其亲和力（根据突变的影响进行了校正）范围为0.02至0.5 muM。单个结构域亲和度的增加倍数从1.1到20不等。亲和度的顺序（从高到低）为4 > 2近似至5 > 6近似至3近似至1。这些结果表明，MTF1的Cys(2)His(2)锌指结构域都没有明显的低金属离子亲和力，当然也没有低到足以响应微摩尔范围内游离锌离子浓度的变化。尽管如此，某些结构域的金属离子亲和性确实相差25倍，在氨基端和羧基端的结构域对金属离子的内在亲和性都比中心结构域低。
Metal response element (MRE) binding transcription factor-1 (MTF1) is a six Cys(2)His(2) zinc finger-containing transcription factor required for basal and zinc-induced transcription of metallothionein,genes. The cobalt(II) and zinc(11) affinities of a protein fragment comprising the six zinc finger domains have been examined to reveal apparent dissociation constants (for the six domains collectively) of 0.5 +/- 0.2 muM for cobalt(II) and 31 +/- 14 pM for zinc(11). Two approaches have been used to determine the metal ion affinities of the individual domains. First, the six domains have been examined as single domain peptides revealing dissociation constants ranging from 0.3 to 1.7 muM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (K-d(Co(It)) 0.3-0.5 muM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (K-d(Co(II)) 1.6-1.7 muM). Second, we examined the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(IT) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (corrected for the effect of the mutation) ranging from 0.02 to 0.5 muM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was observed to be 4 > 2 approximate to 5 > 6 approximate to 3 approximate to 1. These results reveal that none of the Cys(2)His(2) zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concentrations in the micromolar range. I Nonetheless, the metal ion affinities of some domains do differ by a factor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains.