Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor.
Structure and function of the Zn(II) binding site within the DNA-binding domain of the GAL4 transcription factor.
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DOI：
10.1016/0162-0134(89)84572-6
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发表时间：
1989-05
影响因子：
11.1
通讯作者：
Tao Pan;JosephE. Coleman
中科院分区：
文献类型：
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作者：
Tao Pan;JosephE. Coleman
酿酒酵母转录因子GAL4在其DNA结合区含有一个类似锌指的基序：Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+-Cys。克隆载体[记为GAL4(149*)]中的GAL4片段由1-147个残基和两个额外残基组成，并在大肠杆菌中高效表达。该片段包括整个DNA结合域(残基1-74)。均一的GAL4-(149*)蛋白质每摩尔蛋白质含有1-1.5摩尔的锌(II)。GAL4(149*)蛋白与GAL4结合位点上发现的特定的17碱基对回文DNA序列紧密结合，通过使用包含该序列的32P标记的23-聚体的凝胶保留试验显示。EDTA在低pH条件下去除本征锌离子，取消了与23-聚体的结合。GAL4(149*)脱辅基蛋白可与锌(II)、镉(II)或钴(II)重组，恢复与DNA的特异性结合。用~(113)Cd(II)滴定GAL4(149*)载脂蛋白，发现分子上有两个~(113)Cd(II)结合部位，其中一个与四个硫原子配位，一个与四个硫原子配位，另一个与三个或四个硫原子配位。由于Gal4(149*)蛋白在其DNA结合区仅含有6个半胱氨酸残基，因此不能确定两个Cd(II)离子的精确配位；可能存在一个或多个共享的S配体。根据圆二色谱估计，GAL4(149*)蛋白含有大约40%的α-螺旋和大约20%的β-折叠。去除天然的锌离子会导致二级结构的有限展开，但不到一圈的α-螺旋。锌(II)、镉(II)和钴(II)与GAL4(149*)脱辅基蛋白的结合保护蛋白质不被胰酶降解，从而产生一个13 kDa的DNA结合核心。
The transcription factor GAL4 from Saccharomyces cerevisiae contains a "zinc-finger"-like motif, Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys, within its DNA-binding domain. A GAL4 fragment consisting of residues 1-147 plus two additional residues from the cloning vector [denoted GAL4(149*)] has been cloned and overexpressed in Escherichia coli. This fragment includes the entire DNA-binding domain (residues 1-74). The homogeneous GAL4-(149*) protein contains 1-1.5 moles of Zn(II) per mole of protein. The GAL4(149*) protein binds tightly to the specific 17-base-pair palindromic DNA sequence found at GAL4 binding sites as shown by gel-retention assays using a 32P-labeled 23-mer containing this sequence. Removal of the intrinsic Zn(II) by EDTA at low pH abolishes binding to the 23-mer. The GAL4(149*) apoprotein can be reconstituted with Zn(II), Cd(II), or Co(II) with restoration of specific DNA binding. Titration of GAL4(149*) apoprotein with 113Cd(II) shows two 113Cd(II) binding sites on the molecule, one with delta of 707 ppm, suggesting coordination to four sulfur atoms, and one with delta of 669 ppm, suggesting coordination to three or four sulfur atoms. Because GAL4(149*) protein contains only six cysteine residues within its DNA-binding domain, the precise coordination of the two Cd(II) ions cannot be stated with certainty; one or more shared -S- ligands could exist. GAL4(149*) protein contains approximately 40% alpha-helix and approximately 20% beta-sheet, estimated from circular dichroism. Removal of the native Zn(II) ion causes limited unfolding of secondary structure, but less than one turn of alpha-helix. The binding of Zn(II), Cd(II), and, to a lesser extent, Co(II) to GAL4(149*) apoprotein protects the protein from proteolysis by trypsin, which produces a 13-kDa DNA-binding core.