Pan T, Coleman J E
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510.
Proc Natl Acad Sci U S A. 1989 May;86(9):3145-9. doi: 10.1073/pnas.86.9.3145.
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.
来自酿酒酵母的转录因子GAL4在其DNA结合结构域中含有一个“锌指”样基序，即Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6 +++-Cys。由1-147位残基加上来自克隆载体的另外两个残基组成的GAL4片段[表示为GAL4(149*)]已被克隆并在大肠杆菌中过表达。该片段包括整个DNA结合结构域（1-74位残基）。均一的GAL4-(149*)蛋白每摩尔蛋白含有1-1.5摩尔的Zn(II)。如使用包含该序列的32P标记的23聚体进行凝胶滞留试验所示，GAL4(149*)蛋白与在GAL4结合位点发现的特定17碱基对回文DNA序列紧密结合。在低pH下用EDTA去除内在的Zn(II)会消除与23聚体的结合。GAL4(149*)脱辅基蛋白可以用Zn(II)、Cd(II)或Co(II)进行重构，并恢复特异性DNA结合。用113Cd(II)对GAL4(149*)脱辅基蛋白进行滴定显示分子上有两个113Cd(II)结合位点，一个δ为707 ppm，表明与四个硫原子配位，另一个δ为669 ppm，表明与三个或四个硫原子配位。由于GAL4(149*)蛋白在其DNA结合结构域中仅含有六个半胱氨酸残基，因此无法确定两个Cd(II)离子的确切配位情况；可能存在一个或多个共享的-S-配体。根据圆二色性估计，GAL4(149*)蛋白含有约40%的α-螺旋和约20%的β-折叠。去除天然的Zn(II)离子会导致二级结构有限展开，但α-螺旋展开不到一圈。Zn(II)、Cd(II)以及程度较轻的Co(II)与GAL4(149*)脱辅基蛋白的结合可保护该蛋白免受胰蛋白酶的蛋白水解作用，胰蛋白酶会产生一个13 kDa的DNA结合核心。
