Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454, United States.
Biochemistry. 2011 Feb 8;50(5):788-94. doi: 10.1021/bi1019258. Epub 2011 Jan 11.
X-ray crystal structures have been previously determined for three CLC-type transporter homologues, but the absolute unitary transport rate is known for only one of these. The Escherichia coli Cl(-)/H(+) antiporter (EC) moves ∼2000 Cl(-) ions/s, an exceptionally high rate among membrane-transport proteins. It is not known whether such rapid turnover is characteristic of ClCs in general or if the E. coli homologue represents a functional outlier. Here, we characterize a CLC Cl(-)/H(+) antiporter from the cyanobacterium Synechocystis sp. PCC6803 (SY) and determine its crystal structure at 3.2 Å resolution. The structure of SY is nearly identical to that of EC, with all residues involved in Cl(-) binding and proton coupling structurally similar to their equivalents in EC. SY actively pumps protons into liposomes against a gradient and moves Cl(-) at ∼20 s(-1), 1% of the EC rate. Electrostatic calculations, used to identify residues contributing to ion binding energetics in SY and EC, highlight two residues flanking the external binding site that are destabilizing for Cl(-) binding in SY and stabilizing in EC. Mutation of these two residues in SY to their counterparts in EC accelerates transport to ∼150 s(-1), allowing measurement of Cl(-)/H(+) stoichiometry of 2/1. SY thus shares a similar structure and a common transport mechanism to EC, but it is by comparison slow, a result that refutes the idea that the transport mechanism of CLCs leads to intrinsically high rates.
先前已经确定了三种 CLC 型转运蛋白同源物的 X 射线晶体结构，但这些结构中只有一种的单位转运速率是已知的。大肠杆菌 Cl(-)/H(+)反向转运蛋白（EC）的转运速度约为 2000 个 Cl(-)离子/s，在膜转运蛋白中属于极高的速率。目前尚不清楚这种快速周转率是否是 ClCs 的普遍特征，或者大肠杆菌同源物是否代表了一种功能异常。在这里，我们对来自蓝藻集胞藻 PCC6803（SY）的 CLC Cl(-)/H(+)反向转运蛋白进行了表征，并确定了其在 3.2 Å分辨率下的晶体结构。SY 的结构与 EC 的结构几乎完全相同，所有参与 Cl(-)结合和质子偶联的残基在结构上与 EC 中的等效残基相似。SY 主动将质子泵入脂质体中以形成梯度，并以约 20 s(-1)的速度转运 Cl(-)，这一速度是 EC 速率的 1%。静电计算用于鉴定在 SY 和 EC 中参与离子结合能的残基，突出了位于外部结合位点两侧的两个残基，它们在 SY 中不利于 Cl(-)结合，而在 EC 中有利于 Cl(-)结合。将 SY 中的这两个残基突变为 EC 中的相应残基，可将转运速度加速至约 150 s(-1)，从而可以测量 Cl(-)/H(+)的转运比为 2/1。因此，SY 与 EC 具有相似的结构和共同的转运机制，但与之相比，SY 的转运速度较慢，这一结果驳斥了 CLCs 的转运机制导致内在高速度的观点。
