Structure/Function of Mitochondrial Citrate Carrier

描述（由申请人提供）：本项目的长期目标是了解线粒体柠檬酸盐转运蛋白（CTP）的分子结构与其转运机制之间的关系。这种转运蛋白催化三羧酸、二羧酸和磷酸烯醇丙酮酸穿过线粒体内膜的交换，因此对真核细胞的能量代谢至关重要。最近，我们：i）进行跨膜结构域（TMD）Hl和IV的半胱氨酸扫描诱变研究，其与化学修饰、氮氧化物扫描和底物保护实验组合，允许鉴定底物易位途径的必要部分;和iii）开发了在结晶相容的洗涤剂中纯化CTP的方法，这使得能够开始全面的结晶试验。在此基础上，我们建议开展研究，继续推进我们对这种代谢重要转运蛋白功能的理解。具体地，将进行实验以：i）确定四种剩余TMD在CTP底物易位途径形成中的贡献（通过在基于我们的同源性建模的CTP结构选择的位置处的半胱氨酸取代诱变，然后对单个Cys突变体进行化学修饰）并鉴定形成静电漏斗的残基，所述静电漏斗将柠檬酸盐从其表面吸引到途径中; iii）鉴定形成同源二聚体CTP中的两个CTP单体之间的界面的残基，并使用定点自旋标记和硫醇交联表征在转运期间发生的配体诱导的构象变化;和iv）确定能够生长X射线衍射质量的CTP晶体的条件，然后测定CTP结构。这些研究将为线粒体CTP功能的化学和结构基础提供全面的了解。该项目的健康相关性涉及CTP在生物能量学中的核心作用。因此，疾病中CTP功能的改变（例如，糖尿病、癌症）是表征这些病理的异常代谢的重要方面。因此，阐明通过CTP的底物运输的结构基础对于理解CTP在正常和病理状态下的能量产生中的作用是至关重要的。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the relationship between the molecular structure of the mitochondrial citrate transport protein (CTP) and its mechanism of transport. This transporter catalyzes the exchange of tricarboxylates, dicarboxylates, and phosphoenolpyruvate across the inner mitochondrial membrane, and as such is essential to the energy metabolism of eukaryotic cells. Recently, we: i) conducted cysteine scanning mutagenesis studies of transmembrane domains (TMDs) HI and IV which, in combination with chemical modification, nitroxide scanning, and substrate protection experiments, permitted identification of essential portions of the substrate translocation pathway; ii) developed a homology model of the CTP structure; and iii) developed methods for the purification of the CTP in crystallization-compatible detergents, which enabled the initiation of comprehensive crystallization trials. From this foundation, we propose to launch studies that will continue the fundamental advancement in our understanding of the functioning of this metabolically important transporter. Specifically, experiments will be conducted to: i) define the contributions of the four remaining TMDs in the formation of the CTP substrate translocation pathway (via cysteine-substitution mutagenesis at locations chosen on the basis of our homology modeled CTP structure followed by chemical modification of the single Cys mutants) and identify residues forming an electrostatic funnel that attracts citrate into the pathway from its surfaces; ii) identify the substrate binding site(s) within the translocation pathway and assess the ability of selected CTP domains to control substrate access to the pathway; iii) identify residues forming the interface between two CTP monomers in homodimeric CTP and characterize the ligand-induced conformational changes that occur during transport using site-directed spin labeling and thiol cross-linking; and iv) identify conditions enabling the growth of X-ray diffraction quality CTP crystals followed by determination of the CTP structure. These studies will provide a comprehensive understanding of the chemical and structural bases for mitochondrial CTP function. The health relatedness of this project concerns the central role of the CTP in bioenergetics. Thus, altered CTP function in disease (e.g., diabetes, cancer) is an important aspect of the aberrant metabolism that characterizes these pathologies. Consequently, an elucidation of the structural basis for substrate transport through the CTP is critical to understanding the CTP's role in energy production in normal and pathological states.