Department of Biochemistry & Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064, USA.
J Bioenerg Biomembr. 2010 Apr;42(2):99-109. doi: 10.1007/s10863-010-9280-0. Epub 2010 Mar 31.
The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter's two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5'-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.
本研究利用电子顺磁共振（EPR）光谱的定点自旋标记方法，鉴定了柠檬酸（天然配体）和转运抑制剂对重新构建在脂质体囊泡中的酵母线粒体柠檬酸转运蛋白（CTP）构象的影响。在 CTP 的六个不同位置放置了自旋标记，以监测发生在每个转运体的两个底物结合位点附近以及 CTP 结构内更远的结构域的构象变化。我们观察到，柠檬酸对 EPR 谱几乎没有影响。相比之下，转运抑制剂 1,2,3-苯三甲酸（BTC）、吡哆醛 5'-磷酸（PLP）和化合物 792949 导致的光谱变化表明，在六个测试位置中的每个位置，附着的自旋标记的灵活性降低。固定化效应的顺序为化合物 792949 > PLP > BTC。四个报告 CTP 底物结合位点的自旋标记位置在加入抑制剂后，EPR 光谱发生了最大的变化。此外，我们发现当化合物 792949 向量添加（即额外和/或内脂质体）时，固定化效应主要由外部试剂介导。相比之下，当向量添加 PLP 时，外部和内部隔室都以相似的程度介导了该效应。综合我们的数据表明：i）柠檬酸与 CTP 底物结合位点的结合不会以全局方式改变侧链和/或骨架的流动性，这与我们的预期一致，即在没有和存在底物的情况下，CTP 表现出膜转运体所需的灵活性；ii）测试的每种转运抑制剂的结合将多个 CTP 结构域锁定在更刚性的构象中，从而表现出长程结构域间构象通讯。化合物 792949 和 PLP 的差异向量效应在 CTP 同源建模结构的背景下进行了讨论，并给出了潜在的机制分子解释。
