Thorvaldsen J L, Sewell A K, Tanner A M, Peltier J M, Pickering I J, George G N, Winge D R
Department of Medicine, University of Utah Health Science Center, Salt Lake City 84132.
Biochemistry. 1994 Aug 16;33(32):9566-77. doi: 10.1021/bi00198a024.
AMT1 is the transcription factor required for Cu-induced expression of metallothionein genes in the yeast Candida glabrata. The copper-binding, DNA-binding domain of AMT1 has been purified after expression of an AMT1 synthetic gene in bacteria and was confirmed as active in a gel shift assay. The Cu-activated AMT1 was shown to contain a Cu(+)-thiolate tetracopper center and a single Zn2+ site. AMT1 is purified as a Cu-Zn protein from bacterial cultures grown in the presence of CuSO4. Chemical analysis suggested that 4.2 +/- 0.2 and 1.2 +/- 0.2 molar equiv copper and zinc ions bound, respectively. Electrospray mass spectrometry was used to verify that a uniform species was present with 4 Cu+ ions and 1 Zn2+ ion bound per AMT1 molecule. Cu+ binding to form a tetracopper center occurs cooperatively as shown by electrospray MS of apoAMT1 samples reconstituted with increasing equivalency of Cu+. Copper-thiolate coordination was indicated by Cu-S charge-transfer transitions in the ultraviolet, luminescence typical of Cu-thiolate clusters and EXAFS. Analysis of the EXAFS of CuZnAMT1 revealed predominantly trigonal Cu+ coordination and the presence of a polycopper cluster by virtue of a short Cu-Cu distance of 2.7 A. Zn K-edge EXAFS of Cu4Zn1AMT1 and electronic spectroscopy of AMT1 with Co2+ substituted for the single Zn2+ ion are consistent with tetrahedral Zn2+ coordination with thiolate ligands. The Cu-activated AMT1 exhibited a conformation distinct from that of metal-free AMT1 as shown by circular dichroism. DNA binding by AMT1 was dependent on the tetracopper center but was independent of occupancy of the Zn2+ site. This is the first report of a single, uniform tetracopper center in a metal-activated transcription factor.
AMT1是铜诱导光滑念珠菌中金属硫蛋白基因表达所需的转录因子。在细菌中表达AMT1合成基因后，已纯化出AMT1的铜结合、DNA结合结构域，并在凝胶迁移试验中证实其具有活性。铜激活的AMT1显示含有一个Cu(+) -硫醇盐四铜中心和一个单一的Zn2+位点。AMT1是从在硫酸铜存在下生长的细菌培养物中作为铜锌蛋白纯化得到的。化学分析表明，分别结合了4.2±0.2和1.2±0.2摩尔当量的铜离子和锌离子。电喷雾质谱用于验证每个AMT1分子结合4个Cu+离子和1个Zn2+离子时存在单一均匀的物种。如用增加当量的Cu+重构的脱辅基AMT1样品的电喷雾质谱所示，Cu+结合形成四铜中心是协同发生的。紫外光下的Cu-S电荷转移跃迁、Cu-硫醇盐簇典型的发光以及扩展X射线吸收精细结构（EXAFS）表明存在铜-硫醇盐配位。对CuZnAMT1的EXAFS分析显示主要是三角Cu+配位，并且由于短的Cu-Cu距离2.7 Å而存在多铜簇。Cu4Zn1AMT1的Zn K边EXAFS以及用Co2+取代单一Zn2+离子的AMT1的电子光谱与Zn2+与硫醇盐配体的四面体配位一致。如圆二色性所示，铜激活的AMT1呈现出与无金属AMT1不同的构象。AMT1与DNA的结合依赖于四铜中心，但与Zn2+位点的占据无关。这是关于金属激活转录因子中单一均匀四铜中心的首次报道。
