X-Ray Structure and Site-Directed Mutagenesis Analysis of the Escherichia coli Colicin M Immunity Protein
X-Ray Structure and Site-Directed Mutagenesis Analysis of the Escherichia coli Colicin M Immunity Protein
复制标题
DOI：
10.1128/jb.01119-10
复制
发表时间：
2011-01-01
影响因子：
3.2
通讯作者：
Mengin-Lecreulx, Dominique
中科院分区：
文献类型：
--
作者：
Gerard, Fabien;Brooks, Mark A.;Mengin-Lecreulx, Dominique
大肠杆菌素M （Colicin M, ColM）是由一些大肠杆菌菌株产生的，用于杀死来自相同或相关物种的竞争菌株，最近被证明可以通过酶降解其脂质II前体来抑制细胞壁肽聚糖的生物合成。产生colm的菌株通过一种名为Cmi的特定免疫蛋白的共表达而免受其产生的毒素的侵害，Cmi的作用方式仍有待确定。本文报道了由4条β链和4条α螺旋组成的Cmi晶体结构的分辨率。这个相当紧凑的结构揭示了残基Cys31和Cys107之间的二硫键。有趣的是，这两个半胱氨酸和其他几个残基在属于YebF家族的几个功能未知的蛋白质序列中似乎是保守的，这些蛋白质与Cmi的总体序列相似性为25%至35%。通过位点定向诱变来评估这些残基在Cmi的ColM免疫活性中的作用，结果表明，蛋白质c末端的二硫键和残基在功能上是必需的。还证明了DsbA氧化酶参与Cmi二硫键的形成。
Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four beta strands and four alpha helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated.