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首页> 外文期刊>MicrobiologyOpen >Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae
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Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae

机译：肺炎链球菌介导PcsB水解肽聚糖水解的必需细胞分裂蛋白FtsX和FtsE的生化特性

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Abstract The FtsEX:PcsB complex forms a molecular machine that carries out peptidoglycan (PG) hydrolysis during normal cell division of the major respiratory pathogenic bacterium, Streptococcus pneumoniae (pneumococcus). FtsX is an integral membrane protein and FtsE is a cytoplasmic ATPase that together structurally resemble ABC transporters. Instead of transport, FtsEX transduces signals from the cell division apparatus to stimulate PG hydrolysis by PcsB, which interacts with extracellular domains of FtsX. Structural studies of PcsB and one extracellular domain of FtsX have recently appeared, but little is known about the biochemical properties of the FtsE ATPase or the intact FtsX transducer protein. We report here purifications and characterizations of tagged FtsX and FtsE proteins. Pneumococcal FtsX-GFP-His and FtsX-His could be overexpressed in Escherichia coli without toxicity, and FtsE-His remained soluble during purification. FtsX-His dimerizes in detergent micelles and when reconstituted in phospholipid nanodiscs. FtsE-His binds an ATP analog with an affinity comparable to that of ATPase subunits of ABC transporters, and FtsE-His preparations have a low, detectable ATPase activity. However, attempts to detect complexes of purified FtsX-His, FtsE-His, and PcsB-His or coexpressed tagged FtsX and FtsE were not successful with the constructs and conditions tested so far. In working with nanodiscs, we found that PcsB-His has an affinity for charged phospholipids, mediated partly by interactions with its coiled-coil domain. Together, these findings represent first steps toward reconstituting the FtsEX:PcsB complex biochemically and provide information that may be relevant to the assembly of the complex on the surface of pneumococcal cells.
机译：摘要FtsEX：PcsB复合物形成一种分子机器，该机器在主要呼吸道致病菌肺炎链球菌（肺炎球菌）的正常细胞分裂过程中进行肽聚糖（PG）水解。 FtsX是必不可少的膜蛋白，FtsE是胞质ATPase，在结构上类似于ABC转运蛋白。 FtsEX代替运输，转导来自细胞分裂装置的信号以刺激PcsB水解PG，而PcsB与FtsX的胞外域相互作用。最近出现了对PcsB和FtsX的一个胞外域的结构研究，但对FtsE ATPase或完整的FtsX转化子蛋白的生物化学性质了解甚少。我们在这里报告标记的FtsX和FtsE蛋白的纯化和鉴定。肺炎球菌FtsX-GFP-His和FtsX-His可能在大肠杆菌中过表达而无毒性，并且FtsE-His在纯化过程中仍可溶。 FtsX-His在去污剂胶束中以及在磷脂纳米盘中重构时会二聚。 FtsE-His以与ABC转运蛋白ATPase亚基相当的亲和力与ATP类似物结合，FtsE-His制剂的ATPase活性较低。但是，到目前为止，对检测到的FtsX-His，FtsE-His和PcsB-His或共表达标记的FtsX和FtsE的复合物的尝试均未成功。在处理纳米光盘时，我们发现PcsB-His对带电的磷脂具有亲和力，部分是由与其螺旋线圈结构域的相互作用介导的。总之，这些发现代表了生物化学重建FtsEX：PcsB复合物的第一步，并提供了可能与肺炎球菌细胞表面复合物组装相关的信息。

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