Wang Zhonghua, Feng Linda S, Matskevich Viktor, Venkataraman Krishna, Parasuram Priya, Laity John H
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, USA.
J Mol Biol. 2006 Apr 7;357(4):1167-83. doi: 10.1016/j.jmb.2006.01.010. Epub 2006 Jan 24.
The Zap1 transcription factor controls expression of genes that regulate zinc homeostasis in Saccharomyces cerevisiae. The solution structure of two zinc fingers (zf1-2(CA3)) derived from a zinc-responsive domain of Zap1 (zf1-2) has been determined. Under zinc-limiting conditions, zinc finger 2 (zf2) from this domain has been shown to be a constitutive transcriptional activator. Moreover, repression of zf2 function in zinc-replete cells required zinc coordination to both canonical finger 1 (zf1) and zf2 metal sites, suggesting zf1-zf2 cooperativity underlies Zap1 metalloregulation. A structural basis for this cooperativity is identified here. Favorable inter-helical contacts in zf1-2(CA3) extend the individual finger hydrophobic cores through the zf1-zf2 interface. Tryptophan residues at position 5 in each finger provide numerous non-helical inter-finger contacts reminiscent of those observed in GLI1 zinc fingers 1 and 2. The molecular mechanism for zf1-dependent repression of zf2 transcriptional activation is explored further using NMR and CD titration studies. While zf1 independently forms a betabetaalpha solution structure, the majority of zf2 ensemble solution states do not adopt the canonical betabetaalpha zinc finger fold without zf1-zf2 interactions. Cooperative effects on Zn(II) affinities stemming from these finger-finger interactions are observed also in calorimetric studies, in which the 160(+/-20)nM (zf1) and 250(+/-40)nM (zf2) K(d) values for each individual finger increased substantially in the context of the zf1-2 protein (apparent K(dzf1-2WT)=4.6(+/-1.2)nM). On the basis of the above observations, we propose a mechanism for Zap1 transcriptional regulation in which zf1-zf2 interactions stabilize the betabetaalpha folded "repressed state" of the zf2 activation domain in the presence of cellular Zn(II) excess. Moreover, in contrast to earlier reports of <<1 labile zinc ion/Escherichia coli cell, the zf1-zf2 zinc affinities determined calorimetrically are consistent with Zn(II) levels >>1 labile zinc ion/eukaryotic cell.
Zap1转录因子控制酿酒酵母中调节锌稳态的基因的表达。已确定了源自Zap1锌响应结构域（zf1-2）的两个锌指（zf1-2(CA3)）的溶液结构。在锌限制条件下，该结构域的锌指2（zf2）已被证明是一种组成型转录激活因子。此外，在锌充足的细胞中抑制zf2功能需要锌与典型的锌指1（zf1）和zf2金属位点配位，这表明zf1-zf2协同作用是Zap1金属调节的基础。本文确定了这种协同作用的结构基础。zf1-2(CA3)中有利的螺旋间接触通过zf1-zf2界面扩展了单个锌指的疏水核心。每个锌指第5位的色氨酸残基提供了许多非螺旋的锌指间接触，让人联想到在GLI1锌指1和2中观察到的接触。使用核磁共振和圆二色光谱滴定研究进一步探索了zf1依赖性抑制zf2转录激活的分子机制。虽然zf1独立形成ββα溶液结构，但在没有zf1-zf2相互作用的情况下，大多数zf2整体溶液状态不会采用典型的ββα锌指折叠。在量热研究中也观察到了这些锌指间相互作用对锌（II）亲和力的协同效应，其中在zf1-2蛋白的背景下，每个单独锌指的160（±20）nM（zf1）和250（±40）nM（zf2）的解离常数（K(d)）值大幅增加（表观K(dzf1-2WT)=4.6（±1.2）nM）。基于上述观察结果，我们提出了一种Zap1转录调控机制，即在细胞内锌（II）过量的情况下，zf1-zf2相互作用稳定了zf2激活结构域的ββα折叠“抑制状态”。此外，与早期报道的每个大肠杆菌细胞中锌离子含量小于1个不稳定锌离子不同，通过量热法测定的zf1-zf2锌亲和力与每个真核细胞中锌（II）含量大于1个不稳定锌离子一致。
