Synergism between halide binding and proton transport in a CLC-type exchanger
Synergism between halide binding and proton transport in a CLC-type exchanger
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DOI：
10.1016/j.jmb.2006.07.081
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发表时间：
2006-09-29
影响因子：
5.6
通讯作者：
Dutzler, Raimund
中科院分区：
文献类型：
--
作者：
Accardi, Alessio;Lobet, Severine;Dutzler, Raimund
Cl-/H+交换转运蛋白CLC-ec 1介导两个Cl-离子对一个质子的化学计量跨膜交换。一个保守的酪氨酸离子可见的结构残基，Y 445，协调一个结合的Cl-和这个蛋白质，并位于附近的Cl-和H+途径的交叉点。这种酪氨酸的突变体进行了仔细检查的影响，Cl-和H+确定电生理和蛋白质结构上确定的结晶。尽管Y 445在CLC家族中具有很强的保守性，但在该位置的F或W取代保留了野生型转运行为。然而，被A、E或H取代产生具有稳健的Cl-转运但大大损害H+运动的解偶联蛋白。强制性的2Cl（-）/1 H+化学计量因此在这些突变体中丢失。所有的突变体的结构基本上是相同的野生型，但明显的阴离子占有率在Cl-结合区域与功能H+耦合。特别是，如所确定的晶体生长在Br，电生理（-）主管Cl-类似物的异常衍射，耦合良好的转运显示出强大的Br-结合位点。然而，在电子密度在“内”和“中央”Cl-的解偶联突变体，Br-密度是不存在的中心网站，而仍然存在于内部网站。另一个突变体Y 445 L在功能和结构特征上都是中间型的。该突变体明显地将H+交换为Cl-，但H+与Cl-的比率降低;同样地，中心和内部位点都被Br-占据，但中心位点显示出比野生型（或Y 445 FW）低的Br-密度。质子耦合和中心位点占有率之间的相关性认为，卤化物结合到中心运输位点以某种方式促进了H+的移动，这是一种协同作用，在交替位点反向转运方案方面不容易理解。(c)2006爱思唯尔有限公司版权所有。
The Cl-/H+ exchange-transporter CLC-ec1 mediates stoichiometric transmembrane exchange of two Cl- ions for one proton. A conserved tyrosine ions visible in the structure residue, Y445, coordinates one of the bound Cl- and of this protein and is located near the intersection of the Cl- and H+ pathways. Mutants of this tyrosine were scrutinized for effects on the coupled transport of Cl- and H+ determined electrophysiologically and on protein structure determined crystallographically. Despite the strong conservation of Y445 in the CLC family, substitution of F or W at this position preserves wild-type transport behavior. Substitution by A, E, or H, however, produces uncoupled proteins with robust Cl- transport but greatly impaired movement of H+. The obligatory 2Cl(-) /1 H+ stoichiometry is thus lost in these mutants. The structures of all the mutants are essentially identical to wild-type, but apparent anion occupancy in the Cl- binding region correlates with functional H+ coupling. In particular, as determined by anomalous diffraction in crystals grown in Br, an electrophysiologically (-) competent Cl- analogue, the well-coupled transporters show strong Br- binding sites. However, in electron density at the "Inner" and "central" Cl- the uncoupled mutants, Br- density is absent at the central site, while still present at the inner site. An additional mutant, Y445L, is intermediate in both functional and structural features. This mutant clearly exchanges H+ for Cl-, but at a reduced H+-to-Cl- ratio; likewise, both the central and inner sites are occupied by Br-, but the central site shows lower Br- density than in wild-type (or in Y445FW). The correlation between proton coupling and central-site occupancy argues that halide binding to the central transport site somehow facilitates movement of H+, a synergism that is not readily understood in terms of alternating-site antiport schemes. (c) 2006 Elsevier Ltd. All rights reserved.