Programme of Molecular Structure and Function, Research Institute, the Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 2011 Feb 25;286(8):6733-41. doi: 10.1074/jbc.M110.175877. Epub 2010 Dec 20.
ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation.
ATP 结合增强了 ClC-5 的活性，ClC-5 是 Dent 病（一种影响肾脏近端小管的疾病）中突变的转运蛋白。此前，该膜蛋白胞质区域的 X 射线晶体结构揭示了其 ATP 结合位点。通过突变（Y617A-ClC-5）破坏该位点，减少了全长转运体在非洲爪蟾卵母细胞中的功能性表达和 ATP 依赖性调节。然而，缺乏对 ATP 依赖性调节所涉及的构象变化的深入了解。在这里，我们表明 ATP 结合诱导了蛋白质构象的变化。具体而言，小角度 X 射线散射实验表明，ATP 结合促进了分离的 ClC-5 羧基末端区域的夹状闭合。有限的蛋白水解研究表明，ATP 结合也诱导完整膜蛋白中羧基末端区域的构象紧缩。在成纤维细胞和近端肾小管上皮细胞中，全长 ClC-5（Y617A-ClC-5）中 ATP 结合位点的破坏导致其在高尔基体内的加工和运输缺陷。这些新发现解释了之前报道的这种 ATP 结合突变体功能表达减少的原因，并促使进一步研究一种模型，即 ATP 结合引起的构象紧缩促进了生物合成成熟。
