Strausak Daniel, Howie Michelle K, Firth Stephen D, Schlicksupp Andrea, Pipkorn Rudiger, Multhaup Gerd, Mercer Julian F B
Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood, Vic 3125, Australia.
J Biol Chem. 2003 Jun 6;278(23):20821-7. doi: 10.1074/jbc.M212437200. Epub 2003 Apr 4.
Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.
过量的铜通过门克斯（Menkes）或威尔逊（Wilson）P型ATP酶（分别为MNK和WND）从哺乳动物细胞中流出。MNK和WND在其N端细胞质区域有六个含有CXXC基序的金属结合位点（MBS）。有证据表明，铜由三种细胞质铜伴侣之一的Atox1递送至ATP酶。监测Atox1与MNK的直接相互作用并确定动力学参数的尝试尚未成功。在这里，我们使用两种不同的方法研究了Atox1与MNK的MBS野生型和突变型对的相互作用：酵母双杂交分析和实时表面等离子体共振（SPR）。两种方法均观察到Atox1与MNK的MBS之间存在铜依赖性相互作用。保守的CXXC基序的半胱氨酸（Cys）到丝氨酸（Ser）突变影响了Atox1的结合，突出了半胱氨酸残基对铜诱导相互作用的必要性。尽管酵母双杂交试验未能显示Atox1与MBS5/6的相互作用，但SPR分析清楚地证明了与所有六个MBS的铜依赖性结合，突出了SPR与酵母双杂交系统等其他更间接方法相比的强大功能和灵敏度。所有MBS的铜依赖性伴侣-MBS相互作用的结合常数确定为10-5-10-6 m，代表相对低亲和力的结合事件。Atox1与MBS对的相互作用是非协同的。因此，MNK N端MBS的功能差异不能归因于协同效应或铜伴侣Atox1与MBS的不同亲和力。
