Nguitragool Wang, Miller Christopher
Department of Biochemistry, Howard Hughes Medical Institute and Brandeis University, Waltham, MA 02454, USA.
Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20659-65. doi: 10.1073/pnas.0708639104. Epub 2007 Dec 18.
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(-) 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.
CLC氯离子/氢离子交换体是同二聚体，每个亚基内都含有氯离子结合和氢离子偶联残基。目前尚不清楚运输机制是否需要亚基之间的构象重排，或者每个亚基是否作为一个独立的交换体发挥作用。我们在已知结构的细菌CLC交换体CLC-ec1的无半胱氨酸背景上设计了各种半胱氨酸替代突变体，目的是将亚基共价连接起来。构建体在空气中或与外源性氧化剂进行交联，然后对交联后的蛋白质进行重组以评估其功能。除了传统的二硫键外，还以碘作为氧化剂形成了半胱氨酸-赖氨酸跨桥。所有含有一个、两个或四个跨桥的构建体在氯离子周转率、氯离子/氢离子交换化学计量以及由氯离子梯度驱动的氢离子泵浦方面在功能上是活跃的且动力学上是有效的。这些结果表明，对于CLC通道中已知的“共同门控”所发生的那种大规模四级重排，对于离子运输循环来说并非必要，因此运输机制很可能是由各个亚基独立运作来完成的，就像CLC通道的“快速门控”一样。
