On parallel and antiparallel topology of a homodimeric multidrug transporter
On parallel and antiparallel topology of a homodimeric multidrug transporter
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DOI：
10.1074/jbc.m607186200
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发表时间：
2006-11-24
影响因子：
4.8
通讯作者：
Schuldiner, Shimon
中科院分区：
文献类型：
--
作者：
Soskine, Misha;Mark, Shirley;Schuldiner, Shimon
最近建议的反平行拓扑结构的EmrE离子耦合转运蛋白的生物学的许多方面有有趣的影响。然而，这与生物化学数据不一致，这些数据证明了完整细胞中所有原聚体的相同拓扑结构以及广泛的交联研究。为了研究这一明显的矛盾，我们化学交联二聚体与刚性双官能马来酰亚胺使用半胱氨酸取代位置不允许的反平行拓扑结构。纯化的交联二聚体结合底物，并以与非交联二聚体相似的动力学常数在蛋白脂质体中转运。交联二聚体不与非交联二聚体相互作用，这是根据失活突变体不影响其活性（负显性）这一事实判断的。结果支持的论点，EmrE与并行拓扑结构是完全功能。我们表明，在结晶中使用的洗涤剂增加溶液中的单体的分数。我们认为观察到的反平行取向是晶体中单体排列的结果。EmrE与建议的反平行取向的单体的功能仍有待表征。
The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ion-coupled transporters. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and with extensive cross-linking studies. To examine this apparent contradiction we chemically cross-linked dimers with a rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology. A purified cross-linked dimer binds substrate and transports it in proteoliposomes with kinetic constants similar to those of the non-cross-linked dimer. The cross-linked dimers do not interact with non-cross-linked dimers as judged from the fact that inactive mutants do not affect their activity ( negative dominance). The results support the contention that EmrE with parallel topology is fully functional. We show that the detergents used in crystallization increase the fraction of monomers in solution. We suggest that the antiparallel orientation observed is a result of the arrangement of the monomers in the crystal. Functionality of EmrE with the suggested antiparallel orientation of the monomers remains to be characterized.