Structure/Function of Mitochondrial Citrate Carrier

项目描述（由申请人提供）：该项目的长期目标是了解线粒体柠檬酸转运蛋白（CTP）的分子结构与其转运机制之间的关系。这种转运体催化三羧酸、二羧酸和磷酸烯醇丙酮酸在线粒体内膜上的交换，因此对真核细胞的能量代谢至关重要。最近，我们：i)开展了跨膜结构域（TMDs） HI和IV的半胱氨酸扫描诱变研究，结合化学修饰、氮氧化物扫描和底物保护实验，确定了底物易位途径的重要部分；ii)建立了CTP结构的同源性模型；iii)开发了在结晶兼容洗涤剂中纯化CTP的方法，这使得全面结晶试验的开始成为可能。在此基础上，我们建议开展研究，继续深入了解这种重要代谢转运体的功能。具体来说，实验将进行：i)确定剩余的四个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模型CTP结构选择的位置进行半胱氨酸替代突变，然后对单个Cys突变体进行化学修饰），并确定形成静电漏斗的残基，该漏斗将柠檬酸盐从其表面吸引到该途径中；ii)鉴定易位途径内的底物结合位点，并评估选定的CTP结构域控制底物进入该途径的能力；iii)鉴定同二聚体CTP中形成两个CTP单体之间界面的残基，并利用定向自旋标记和硫醇交联表征在转运过程中发生的配体诱导的构象变化；iv)确定x射线衍射质量CTP晶体生长的条件，然后确定CTP结构。这些研究将提供对线粒体CTP功能的化学和结构基础的全面了解。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（如糖尿病、癌症）中CTP功能的改变是表征这些病理的异常代谢的一个重要方面。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能量产生中的作用至关重要。

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.