Analysis of Structure-Function Relationships in the Colibactin-Maturating Enzyme ClbP
Analysis of Structure-Function Relationships in the Colibactin-Maturating Enzyme ClbP
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DOI：
10.1016/j.jmb.2012.09.017
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发表时间：
2012-12-07
影响因子：
5.6
通讯作者：
Bonnet, Richard
中科院分区：
文献类型：
--
作者：
Cougnoux, Antony;Gibold, Lucie;Bonnet, Richard
大肠杆菌pks基因组岛参与非核糖体肽型基因毒素大肠杆菌蛋白的合成，该基因毒素已被认为影响宿主免疫反应并对癌症发展有影响。pks编码的酶ClbP是一种非典型的肽酶，它有助于大肠杆菌蛋白的合成。在这项工作中，我们确定了ClbP的关键特征。细菌分离和Western-blot分析显示ClbP通过含有三个预测的跨膜螺旋的c端结构域与细菌内膜对接。虽然只需要一个螺旋就可以定位在细胞膜上，但c端结构域的完整序列对于ClbP的生物活性是必要的。此外，ClbP的n端序列允许SRP/Sec/YidC-和mreb依赖性酶结构域在质周室中的易位，这也是ClbP生物活性所必需的特征。最后，将ClbP的结构与FmtA-like和AmpC的结构进行比较，揭示了ClbP在催化槽的一端具有负静电电位的表面特征。定点诱变实验在该区域发现了两个对ClbP生物活性很重要的天冬氨酸残基。总的来说，这些结果提出了ClbP激活前大肠杆菌蛋白的模型，并为设计针对大肠杆菌蛋白产生的抑制剂铺平了道路。(C) 2012 Elsevier Ltd.版权所有。
pks genomic island of Escherichia coli is involved in the synthesis of the non-ribosomal peptide-type genotoxin colibactin, which has been suggesting as affecting the host immune response and having an impact on cancer development. The pks-encoded enzyme ClbP is an atypical peptidase that contributes to the synthesis of colibactin. In this work, we identified key features of ClbP. Bacterial fractionation and Western-blot analysis revealed the docking of ClbP to the bacterial inner membrane via a C-terminal domain harboring three predicted transmembrane helices. Whereas only one helix was necessary for the location in the inner membrane, the complete sequence of the C-terminal domain was necessary for ClbP bioactivity. In addition, the N-terminal sequence of ClbP allowed the SRP/Sec/YidC- and MreB-dependent translocation of the enzymatic domain in the periplasmic compartment, a feature also essential for ClbP bioactivity. Finally, the comparison of ClbP structure with that of the paralogs FmtA-like and AmpC revealed at an extremity of the catalytic groove a negative electrostatic potential surface characteristic of ClbP. Site-directed mutagenesis experiments identified in this zone two aspartic residues that were important for ClbP bioactivity. Overall, these results suggest a model for precolibactin activation by ClbP and pave a way for the design of inhibitors targeting colibactin production. (C) 2012 Elsevier Ltd. All rights reserved.