A periplasmic coiled-coil interface underlying ToIC recruitment and the assembly of bacterial drug eff lux pumps
A periplasmic coiled-coil interface underlying ToIC recruitment and the assembly of bacterial drug eff lux pumps
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DOI：
10.1073/pnas.0610160104
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发表时间：
2007-03-13
影响因子：
11.1
通讯作者：
Koronakis, Vassilis
中科院分区：
文献类型：
--
作者：
Lobedanz, Sune;Bokma, Evert;Koronakis, Vassilis
细菌如大肠杆菌和铜绿假单胞菌通过跨越内膜和外膜以及介于其间的周质空间的三重多药外排泵排出抗生素和其他抑制剂。泵组装中的关键事件是通过同源内膜移位酶的衔接蛋白募集外膜锚定的TolC出口管，建立连续的跨包膜流出孔。我们描述了大肠杆菌广泛分布的RND型泵TolC/AcrAB中并置的周质出口管和接头卷曲螺旋的潜在相互作用。大肠杆菌，使用在体内交联映射的分子间接触的程度。位点特异性TolC半胱氨酸变体与野生型AcrA衔接子的交联鉴定了TolC的下α-螺旋桶结构域上的残基，限定了靠近出口管的入口孔的连续簇。反过来，AcrA半胱氨酸变体与野生型TolC的位点特异性交联鉴定了AcrA卷曲螺旋的N-末端α-螺旋内的衔接子上的相互作用表面。实验数据允许数据驱动的对接方法来模拟泵组件中心的相互作用表面。满足所有交联距离约束的最低能量对接模型将适配器放置在由TolC入口螺旋形成的分子内凹槽处，使适配器卷曲螺旋与暴露的TolC外螺旋对齐。该定位的关键特征在于，其允许在过渡到其打开状态期间用于TolC的内部线圈的所提出的移动的空间。
Bacteria such as Escherichia coli and Pseudomonas aeruginosa expel antibiotics and other inhibitors via tripartite multidrug efflux pumps spanning the inner and outer membranes and the intervening periplasmic space. A key event in pump assembly is the recruitment of an outer membrane-anchored TolC exit duct by the adaptor protein of a cognate inner membrane translocase, establishing a contiguous transenvelope efflux pore. We describe the underlying interaction of juxtaposed periplasmic exit duct and adaptor coiled-coils in the widespread RND-type pump TolC/AcrAB of E. coli, using in vivo cross-linking to map the extent of intermolecular contacts. Cross-linking of site-specific TolC cysteine variants to wild-type AcrA adaptor identified residues on the lower a-helical barrel domain of TolC, defining a contiguous cluster close to the entrance aperture of the exit duct. Reciprocally, site-specific cross-linking of AcrA cysteine variants to wild-type TolC identified the interaction surface on the adaptor within the N-terminal a-helix of the AcrA coiled-coil. The experimental data allowed a data-driven docking approach to model the interaction surface central to pump assembly. The lowest energy docked model satisfying all of the cross-link distance constraints places the adaptor at the intramolecular groove formed by the TolC entrance helices, aligning the adaptor coiled-coil with the exposed TolC outer helix. A key feature of this positioning is that it allows space for the proposed movement of the inner coil of TolC during transition to its open state.