Hussain Faiza, Olson John S, Wittung-Stafshede Pernilla
Department of Biochemistry and Cell Biology, Keck Center for Structural Computational Biology, Houston, TX 77251, USA.
Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11158-63. doi: 10.1073/pnas.0802928105. Epub 2008 Aug 6.
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.
目前尚不清楚人类铜（Cu）伴侣蛋白Atox1如何将铜输送到细胞质中威尔逊病和门克斯病蛋白的金属结合结构域。为了开始解决这个问题，我们对野生型Atox1和两个点突变体（Met(10)Ala和Lys(60)Ala）的铜（I）释放进行了表征。通过使用铜（I）螯合剂二喹啉甲酸（BCA）作为金属受体来测量铜（I）从全Atox1中的置换动力学。BCA通过三步过程从Atox1中去除铜（I），该过程涉及初始Atox1-Cu-BCA复合物的双分子形成，随后Atox1解离以及第二个BCA的结合以生成脱辅基Atox1和Cu-BCA(2)。由于第二个BCA能更快、更轻松地将蛋白质从Atox1-Cu-BCA中间体中置换出来，这两个突变体比野生型Atox1更容易失去铜（I）。值得注意的是，BCA从溶液中摄取铜（I）的速度比从全Atox1中转移的速度要慢得多，这可能是因为DTT-Cu复合物的解离速度较慢。这些结果表明，铜伴侣蛋白在使铜（I）快速被底物利用方面起着关键作用，并且活化的蛋白质-金属-螯合剂复合物可能在动力学上模拟了体内参与铜（I）转移的三元伴侣蛋白-金属-靶标复合物。
