Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach.
Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach.
复制标题
通过定点自旋标记方法探讨转运抑制剂对线粒体柠檬酸转运蛋白构象的影响。
DOI：
10.1007/s10863-010-9280-0
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发表时间：
2010
影响因子：
3
通讯作者：
Kaplan,RonaldS
中科院分区：
文献类型：
--
作者：
Mayor,JuneA;Sun,Jiakang;Kotaria,Rusudan;Walters,DEric;Oh,KyoungJoon;Kaplan,RonaldS
本调查利用电子顺磁共振（EPR）光谱的定点自旋标记方法，以确定柠檬酸盐，天然配体，和运输抑制剂的构象上的酵母线粒体柠檬酸盐转运蛋白（CTP）在脂质体囊泡重建的效果。将自旋标记物放置在CTP内的六个不同位置处，以监测转运蛋白的两个底物结合位点中的每一个附近以及CTP架构内的更远结构域处发生的构象变化。我们观察到，柠檬酸盐引起的EPR谱的变化不大。相比之下，转运抑制剂1，2，3-苯三甲酸酯（BTC）、吡哆醛5′-磷酸盐（PLP）和化合物792949导致光谱变化，表明在测试的六个位置中的每一个位置处附着的自旋标记的柔性降低。固定效果的顺序为化合物792949 > PLP > BTC。CTP底物结合位点上报告的四个自旋标记位置在添加抑制剂后显示EPR谱的最大变化。此外，我们发现当化合物792949以矢量方式添加时（即，脂质体外和/或脂质体内），固定作用几乎完全由外部试剂介导。相比之下，在载体上添加PLP后，从外部和内部隔室介导的效果达到相似的程度。i）柠檬酸盐与CTP底物结合位点的结合不会以整体方式改变侧链和/或主链的移动性，并且与我们的预期一致，即在底物不存在和存在的情况下，CTP都显示出膜转运蛋白所需的柔性;和ii）所测试的每种转运抑制剂的结合将多个CTP结构域锁定为更刚性的构象，从而表现出长程结构域间构象通讯。化合物792949和PLP的差分矢量效应进行了讨论的CTP同源建模的结构和潜在的机制分子的解释。
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.