Yang Wu, Shah Aabid Manzoor, Dong Shiqi, Sun Caili, Zhang Huaiyuan, Mohamed Hassan, Gao Xiuzhen, Fan Huirong, Song Yuanda
Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Sciences, Shandong University of Technology, Zibo, China.
Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
Front Nutr. 2021 Dec 9;8:802231. doi: 10.3389/fnut.2021.802231. eCollection 2021.
The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in , isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the overexpression and knockout plasmids were created and transformed into WJ11. The mitochondria of the -overexpressing transformant of WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the -knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of -4.87 kcal/mol to citric acid, while -3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of at the molecular level and its role in lipid accumulation.
柠檬酸转运蛋白（CTP）在柠檬酸从线粒体基质外流到细胞质的过程中起着重要作用，这在产油真菌中至关重要。柠檬酸外流后产生的细胞质柠檬酸是三酰甘油和胆固醇生物合成途径的主要碳源。由于CTP的重要性，我们实验室对其结构/功能关系进行了广泛研究，以了解其分子机制。在本研究中，已克隆、过表达、纯化、进行动力学和结构表征了WJ11的三羧酸柠檬酸转运蛋白（Tct）。WJ11的Tct蛋白在大肠杆菌中表达、分离，并在脂质体系统中进行功能重组以进行动力学研究。我们的结果表明，Tct对柠檬酸具有高亲和力，Km为0.018 mM。此外，构建了过表达和敲除质粒并将其转化到WJ11中。与野生型WJ11的线粒体相比，WJ11过表达转化体的线粒体柠檬酸外流增加了49%，而敲除转化体的线粒体柠檬酸外流减少了39%。为了阐明这种生物学上重要的转运蛋白的结构-功能关系，使用同源建模构建了线粒体Tct蛋白的三维模型。该蛋白的整体结构为V形，其三维结构为二聚体。还通过分子动力学模拟研究评估了该结构的运输稳定性。对接时，活性结构域被确定与柠檬酸和苹果酸形成氢键和堆积相互作用。三羧酸柠檬酸转运蛋白对柠檬酸的结合能为-4.87 kcal/mol，对苹果酸的结合能为-3.80 kcal/mol。这是首次报道揭示WJ11线粒体Tct蛋白的结构特征以及了解其向底物转运的方式。总之，本研究结果支持我们结合功能和结构数据以在分子水平上更好地理解WJ11的Tct及其在脂质积累中的作用的努力。
