Conserved residues modulate copper release in human copper chaperone Atox1
Conserved residues modulate copper release in human copper chaperone Atox1
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DOI：
10.1073/pnas.0802928105
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发表时间：
2008-08-12
影响因子：
11.1
通讯作者：
Wittung-Stafshede, Pernilla
中科院分区：
文献类型：
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作者：
Hussain, Faiza;Olson, John S.;Wittung-Stafshede, Pernilla
目前还不清楚人类铜（Cu）伴侣Atox 1如何将Cu传递到细胞质中Wilson和Menkes病蛋白的金属结合域。为了开始解决这个问题，我们已经表征了Cu（I）从野生型Atox 1和两个点突变体（Met（10）Ala和Lys（60）Ala）的释放。以Cu（I）螯合剂bicinchonic acid（BCA）为金属受体，测定了Cu（I）从holo-Atox 1上的置换动力学. BCA通过三步过程从Atox 1中去除Cu（I），包括初始Atox 1-Cu-BCA复合物的双分子形成，随后Atox 1解离，第二个BCA结合，生成apo-Atox 1和Cu-BCA（2）。这两种突变体比野生型Atox 1更容易失去Cu（I），因为第二BCA更快速和容易地从Atox 1-Cu-BCA中间体置换蛋白质。值得注意的是，BCA从溶液中吸收Cu（I）比从holo-Atox 1转移慢得多，这可能是因为DTT-Cu络合物的缓慢解离。这些结果表明，Cu分子伴侣在使Cu（I）快速接近底物中起着关键作用，并且活化的蛋白质-金属-螯合剂复合物可以在动力学上模拟参与Cu（I）体内转移的三元分子伴侣-金属-靶复合物。
It is unclear how the human copper (Cu) chaperone Atox1 delivers Cu to metal-binding domains of Wilson and Menkes disease proteins in the cytoplasm. To begin to address this problem, we have characterized Cu(I) release from wild-type Atox1 and two point mutants (Met(10)Ala and Lys(60)Ala). The dynamics of Cu(I) displacement from holo-Atox1 were measured by using the Cu(I) chelator bicinchonic acid (BCA) as a metal acceptor. BCA removes Cu(I) from Atox1 in a three-step process involving the bimolecular formation of an initial Atox1-Cu-BCA complex followed by dissociation of Atox1 and the binding of a second BCA to generate apo-Atox1 and Cu-BCA(2). Both mutants lose Cu(I) more readily than wild-type Atox1 because of more rapid and facile displacement of the protein from the Atox1-Cu-BCA intermediate by the second BCA. Remarkably, Cu(I) uptake from solution by BCA is much slower than the transfer from holo-Atox1, presumably because of slow dissociation of DTT-Cu complexes. These results suggest that Cu chaperones play a key role in making Cu(I) rapidly accessible to substrates and that the activated protein-metal-chelator complex may kinetically mimic the ternary chaperone-metal-target complex involved in Cu(I) transfer in vivo.