Chen Xiaohua, Hua Haiqing, Balamurugan Kuppusamy, Kong Xiangming, Zhang Limei, George Graham N, Georgiev Oleg, Schaffner Walter, Giedroc David P
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Nucleic Acids Res. 2008 May;36(9):3128-38. doi: 10.1093/nar/gkn103. Epub 2008 Apr 13.
Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence (547)CNCTNCKCDQTKSCHGGDC(565) are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Delta strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu(4)-S(6)] cage structure, characterized by a core of trigonally S(3) coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu(4)-L(6) (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I).
黑腹果蝇MTF-1（dMTF-1）是一种铜响应转录激活因子，介导对铜以及锌和镉的抗性。在此，我们鉴定了一个新的富含半胱氨酸的结构域，它对于dMTF-1感知细胞内过量铜至关重要。表达突变型dMTF-1的转基因果蝇，其序列（547）CNCTNCKCDQTKSCHGGDC（565）内的两个、四个或六个半胱氨酸残基被丙氨酸取代，在保护果蝇免受铜毒性的能力上显著受损或完全丧失，并且在过量铜刺激下无法上调金属硫蛋白A（MtnA）的表达。相比之下，这些果蝇在铜缺乏时表现出野生型的存活率，这表明半胱氨酸簇结构域仅在dMTF-1感知铜负荷时是必需的。平行研究表明，分离的半胱氨酸簇结构域对于保护铜敏感的酿酒酵母ace1Delta菌株免受铜毒性是必需的。富含半胱氨酸结构域的肽片段与铜（I）的结合驱动了多齿[Cu(4)-S(6)]笼状结构的协同组装，其特征是由桥连硫醇盐配体结合的三角S(3)配位的铜（I）离子核心。虽然这让人联想到酵母铜调节转录因子中的Cu(4)-L(6)（L =配体）四核簇，但缺乏显著的序列同源性与一种特别适合铜（I）的传感策略的趋同进化是一致的。
