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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >CLC CI~- /H~+ transporters constrained by covalent cross-linking
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CLC CI~- /H~+ transporters constrained by covalent cross-linking

机译：CLC CI〜-/ H〜+转运蛋白受共价交联约束

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摘要

CLC Cl~-/H~+ exchangers are homodimers with Cl~--binding and H~+-coupling residues contained within each subunit. It is not known whether the transport mechanism requires conformational rearrangement between subunits or whether each subunit operates as a separate exchanger. We designed various cysteine substitution mutants on a cysteine-less background of CLC-ec1, a bacterial CLC exchanger of known structure, with the aim of covalently linking the subunits. The constructs were cross-linked in air or with exogenous oxidant, and the cross-linked proteins were reconstituted to assess their function. In addition to conventional disulfides, a cysteine-lysine cross-bridge was formed with I_2 as an oxidant. The constructs, all of which contained one, two, or four cross-bridges, were functionally active and kinetically competent with respect to Cl~1 turnover rate, Cl~-/H~+ exchange stoichiometry, and H~+ pumping driven by a Cl~- gradient. These results imply that large quaternary rearrangements, such as those known to occur for "common gating" in CLC channels, are not necessary for the ion transport cycle and that it is therefore likely that the transport mechanism is carried out by the subunits working individually, as with "fast gating" of the CLC channels.
机译：CLC Cl-// H +交换子是同二聚体，每个亚基中都包含Cl--结合和H +偶联残基。尚不清楚运输机制是否需要子单元之间的构象重排，或者每个子单元是否作为单独的交换器运行。我们在已知结构的细菌CLC交换子CLC-ec1的无半胱氨酸背景下设计了各种半胱氨酸替代突变体，目的是共价连接亚基。将构建体在空气中或与外源性氧化剂交联，并重构交联的蛋白质以评估其功能。除了常规的二硫化物，还形成了以I_2为氧化剂的半胱氨酸-赖氨酸横桥。这些构建体全部包含一个，两个或四个跨桥，它们在功能上具有活性并在动力学上具有与Cl〜1转化率，Cl〜-/ H〜+交换化学计量以及由H2 +驱动的H〜+泵送有关的动力学能力。 Cl〜-梯度。这些结果表明，大的四级重排，例如已知用于CLC通道中“公共门控”的四级重排，对于离子传输循环不是必需的，因此传输机制很可能是由单独工作的亚单元完成的，就像CLC通道的“快速门控”一样。

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