Dissecting the dimerization motif of Enterococcus hirae's Zn(II)CopY.
Dissecting the dimerization motif of Enterococcus hirae's Zn(II)CopY.
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剖析海拉肠球菌 Zn(II)CopY 的二聚化基序。
DOI：
10.1007/s00775-012-0919-7
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Dameron,CharlesT
中科院分区：
文献类型：
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作者：
Collins,TylerC;Dameron,CharlesT
hiaeeenterococcus体内铜稳态通路的调控是通过锌金属蛋白Zn(II)CopY的活性进行的，锌金属蛋白Zn(II)CopY是一种Cu(I)响应的二聚体抑制因子（Cobine etal ., biochemical 41:5822 - 5829, 2002）。它的二聚化结构域包含一个c端富含半胱氨酸的金属结合基序，用于与富含脂肪的重复序列相邻的Cu(I)感应，但尚不清楚哪个区域对相互作用贡献最大。为了实现这一目标，将合成的CopY构建体（CDG）与溶解度增强标签融合在一起，从而可以探测脂肪族重复序列和金属结合位点的关键成分的二聚化活性。使用两种独立的方法测试所得到的融合结构。采用体外等温滴定量热法对二聚体亲和性进行了热力学分析。蛋白质片段互补是一种体内技术，可以快速筛选活细胞内的同二聚体和异二聚体复合物。体内和体外研究相结合，除了破译筛选的所有构建体之间的相对二聚体亲和力外，还可以鉴定二聚体和非二聚体的CDG序列。体内技术允许异质二聚体的形成，以测试它们在不同CDG类似物之间形成特定复合物的能力。
The regulation of the copper homeostasis pathway inEnterococcus hiraeis conducted through activity of the zinc metalloprotein Zn(II)CopY, which is a Cu(I)-responsive dimeric repressor (Cobine et al.,Biochemistry41:5822–5829, 2002). Its dimerization domain contains a C-terminal cysteine-rich metal-binding motif used for Cu(I) sensing adjacent to an aliphatic-rich repeating sequence, but it is unclear as to which regions contribute most to the interaction. To accomplish this, a synthetically produced CopY construct (CDG) was fused with solubility enhancement tags so the key components of the elements of the aliphatic repeat and metal-binding site could be probed for their dimerization activity. The resultant fusion constructs were tested using two independent methods. Isothermal titration calorimetry, an in vitro technique, was employed to determine dimer affinity thermodynamically. Protein fragment complementation, an in vivo technique, made it possible to rapidly screen homodimeric and heterodimeric complexes within live cells. The combination of in vivo and in vitro studies enabled the identification of CDG sequences that dimerize and sequences that do not, in addition to deciphering relative dimer affinity between all constructs screened. The in vivo technique allowed the formation of heterodimers to be tested for their ability to form specific complexes between dissimilar CDG analogs.