The zinc coordination site of the bacteriophage Mu translational activator protein, Com.
The zinc coordination site of the bacteriophage Mu translational activator protein, Com.
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噬菌体 Mu 翻译激活蛋白 Com 的锌配位位点。
DOI：
10.1006/jmbi.1995.0178
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
McLendon,G
中科院分区：
文献类型：
--
作者：
Witkowski,RT;Hattman,S;Newman,L;Clark,K;Tierney,DL;Penner-Hahn,J;McLendon,G
噬菌体Mu Com蛋白是一种小的“锌指样”蛋白，其结合com-mom操纵子mRNA中的特定位点并激活mom开放阅读框的翻译。Com含有6个半胱氨酸和5个组氨酸残基，这些残基有可能形成几个替代的锌指样基序。我们已经使用寡核苷酸定点诱变来单独改变这些氨基酸中的每一个（Cys到Ser，和His到Asn或Gln），并测试了各种形式的Com在体内发挥功能的能力。我们观察到四个N-末端半胱氨酸残基（Cys-6、9、26或29）中的任何一个的突变导致Com活性的丧失。Com蛋白需要锌，以折叠成其功能的三级结构，所示的characteristic 1H核磁共振（NMR）化学位移。1H化学位移恢复到无规卷曲值的金属螯合剂EDTA的存在下。用~ 1H NMR研究了化合物的金属结合特异性和热稳定性。我们报告使用113 Cd NMR，1H-113 Cd杂自旋回波差谱HSED和Zn扩展X射线吸收精细结构光谱EXAFS确定锌/蛋白质的化学计量比为1：1和配体环境为四硫醇盐。Com突变体C6 S和C39 S的比较NMR光谱表明，位置6参与锌配位，而位置39不是金属配体。这些研究表明，金属配位，网站Com是一个四个半胱氨酸配合物，涉及残基6，9，26和29。
The bacteriophage Mu Com protein is a small “zinc finger-like” protein that binds a specific site in com-mom operon mRNA and activates translation of the mom open-reading-frame. Com contains six cysteine and five histidine residues that have the potential to form several alternative zinc-finger-like motifs. We have used oligonucleotide site-directed mutagenesis to individually alter each of these amino acids (Cys to Ser, and His to Asn or Gln) and tested the various forms of Com for their ability to function in vivo. We observed that mutation of any one of the four N-terminal cysteine residues (Cys-6, 9, 26 or 29) resulted in loss of Com activity. The Com protein requires zinc in order to fold into its functional tertiary structure, as demonstrated by characteristic1H nuclear magnetic resonance (NMR) chemical shifts.1H chemical shifts revert to random coil values in the presence of the metal chelator EDTA. The metal-binding specificity and thermal stability of Com also has been investigated using1H NMR. We report the use of113Cd NMR,1H-113Cd heteronuclear spin-echo difference spectroscopy HSED and Zn extended X-ray absorption fine structure spectroscopy EXAFS to determine the zinc/protein stoichiometry as 1:1 and the ligand environment as tetrathiolate. Comparative NMR spectra of Com mutants C6S and C39S suggest position 6 is involved in zinc coordination, while position 39 is not metal-liganded. These studies indicate that the metal coordination, site of Com is a four-cysteine complex, involving residues 6, 9, 26 and 29.