Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis
Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis
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DOI：
10.1002/elps.201100541
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发表时间：
2012-04-01
期刊：
影响因子：
2.9
通讯作者：
Broutin, Isabelle
中科院分区：
文献类型：
--
作者：
Ferrandez, Yann;Monlezun, Laura;Broutin, Isabelle
多药耐药性已成为细菌感染治疗中的一个严重问题。一个突出的作用是归因于外源性物质的主动流出的细菌由三方蛋白质机器。由于大肠杆菌TolC/AcrA/AcrB模型系统和相关的铜绿假单胞菌OprM/MexA/MexB获得的X射线结构，药物挤出的机制相当好理解。然而，许多问题仍未解决，特别是外排泵组件的化学计量。基于蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）（Wittig等人，自然保护区2006，1，418428），我们分析了棕榈酰化和非棕榈酰化MexA与同源配偶体OprM三聚体在不同比例和去污剂条件下的结合化学计量。我们发现，β-辛基吡喃葡萄糖苷（β-OG）洗涤剂不适合这种技术。然后我们证明了MexA必须被棕榈酰化以稳定与OprM的复合物形成。最后，我们提供了证据，棕榈酰化的MexA每OprM三聚体的二乘二（2、4、6或更高）结合。
Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X-ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN-PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418428), we analyzed the binding stoichiometry of both palmitylated and non-palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that beta-octyl glucopyranoside (beta-OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM.