Design, function and structure of a monomeric ClC transporter
Design, function and structure of a monomeric ClC transporter
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DOI：
10.1038/nature09556
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发表时间：
2010-12-09
期刊：
影响因子：
64.8
通讯作者：
Miller, Christopher
中科院分区：
文献类型：
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作者：
Robertson, Janice L.;Kolmakova-Partensky, Ludmila;Miller, Christopher
CLC家族的通道和转运蛋白引起无机阴离子的跨膜运动，为各种生物任务服务，从不寻常的产生千瓦脉冲，电鱼用它击昏它们的猎物，到Escheridian-内体，空泡和溶酶体的酸化（1）。最初从板鳃类Cl-通道的单分子研究中推断（2），后来通过细菌Cl-/H+反向转运蛋白的晶体结构证实（3，4），ClC蛋白的同源二聚体结构显然是普遍的。此外，基本的机械，使离子运动通过这些蛋白质的阴离子扩散的通道和离子耦合室，协调Cl-和H+的转运蛋白中的反向运输的水孔完全包含在每个亚基的同源二聚体。细菌C1 C转运蛋白的近正常功能通过二聚体界面的共价交联和多联体人类同源物的行为来限制，这表明转运循环存在于每个亚基内，并且不需要亚基之间的刚体重排（5，6）。然而，这一证据仅是推论性的，因为已知的例子中，膜外CLC结构域的四元重排有助于二聚化调节转运活性（7），我们不能宣布作为确定的“平行途径”的图片，其中同源二聚体由两个singlesubunit转运蛋白独立运作。这种观点的一个强有力的预测是，原则上应该可以获得单体CLC。在这里，我们利用已知的结构的ClC Cl-/H+交换器，ClC-ec 1从大肠杆菌，设计突变体，不稳定的二聚体界面，同时保留结构和运输功能的各个亚基。结果表明，单独的ClC亚基是运输的基本功能单元，跨亚基相互作用是不需要Cl-/H+交换ClC转运蛋白。
Channels and transporters of the ClC family cause the transmembrane movement of inorganic anions in service of a variety of biological tasks, from the unusual-the generation of the kilowatt pulses with which electric fish stun their prey-to the quotidian-the acidification of endosomes, vacuoles and lysosomes(1). The homodimeric architecture of ClC proteins, initially inferred from single-molecule studies of an elasmobranch Cl- channel(2) and later confirmed by crystal structures of bacterial Cl-/H+ antiporters(3,4), is apparently universal. Moreover, the basic machinery that enables ion movement through these proteins-the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl- and H+ antiport in the transporters-are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial ClC transporter straitjacketed by covalent cross-links across the dimer interface and the behaviour of a concatemeric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits(5,6). However, this evidence is only inferential, and because examples are known in which quaternary rearrangements of extramembrane ClC domains that contribute to dimerization modulate transport activity(7), we cannot declare as definitive a 'parallel-pathways' picture in which the homodimer consists of two singlesubunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric ClC. Here we exploit the known structure of a ClC Cl-/H+ exchanger, ClC-ec1 from Escherichia coli, to design mutants that destabilize the dimer interface while preserving both the structure and the transport function of individual subunits. The results demonstrate that the ClC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl-/H+ exchange in ClC transporters.