Cobine Paul A, George Graham N, Jones Christopher E, Wickramasinghe Wasantha A, Solioz Marc, Dameron Charles T
National Research Center for Environmental Toxicology, University of Queensland, Coopers Plains, QLD 4108, Australia.
Biochemistry. 2002 May 7;41(18):5822-9. doi: 10.1021/bi025515c.
Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(I) per CopZ and two copper(I) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(I)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(I)2CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(I)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper, from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(I) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange; a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.
细胞外铜调节平肠球菌CopY阻遏物的DNA结合活性，从而控制由cop操纵子编码的铜稳态基因的表达。CopY具有CxCxxxxCxC金属结合基序。CopZ是一种铜伴侣蛋白，属于以MxCxxC金属结合基序为特征的金属伴侣蛋白家族，它将铜转移到CopY。通过体外金属重组确定了CopZ和CopY的铜结合化学计量。发现化学计量为每个CopZ一个铜（I），每个CopY单体两个铜（I）。X射线吸收研究表明，Cu（I）CopZ中存在二配位和三配位铜的混合物，但Cu（I）2CopY中存在纯三配位铜配位并伴有Cu-Cu相互作用。后者的配位与CopY的CxCxxxxCxC结合基序中紧密双核Cu（I）-硫醇盐核心的形成一致。用2,4-吡啶偶氮间苯二酚监测铜从CopY中置换锌的情况。需要两个铜（I）离子来释放每个CopY单体结合的单个锌（II）离子。CopZ和CopY之间铜转移的特异性取决于静电相互作用。使用螯合剂二乙基二硫代氨基甲酸（DDC）研究了蛋白质的相对铜结合亲和力。这些数据表明CopY对铜的亲和力高于CopZ。然而，这种亲和力差异不是铜交换的唯一因素；转移反应需要两种蛋白质之间基于电荷的相互作用才能进行。CopZ同源物的功能获得性突变证明了伴侣蛋白上四个赖氨酸残基与CopY相互作用的必要性。综上所述，这些结果提示了CopZ和CopY之间铜交换的机制。
