Structure/Function of Mitochondrial Citrate Carrier

描述(申请人提供)：这个项目的长期目标是了解线粒体柠檬酸盐运输蛋白(CTP)的分子结构与其运输机制之间的关系。这种转运蛋白催化三羧酸盐、二羧酸盐和磷酸烯醇式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是必不可少的。最近，我们：i)进行了跨膜结构域(TMD)HI和IV的半胱氨酸扫描突变研究，结合化学修饰、氮氧化物扫描和底物保护实验，能够识别底物易位途径的必要部分；ii)开发了CTP结构的同源模型；iii)开发了结晶兼容洗涤剂中CTP的纯化方法，从而能够启动全面的结晶试验。在此基础上，我们建议开展研究，继续在我们对这种代谢重要转运体的功能的理解方面取得根本性进展。具体地说，将进行以下实验：i)确定其余四个TMD在形成CTP底物易位途径中的作用(通过半胱氨酸取代突变，在基于我们的同源模拟CTP结构的基础上选择位置，然后对单个半胱氨酸突变体进行化学修饰)，并确定形成吸引柠檬酸从其表面进入该途径的静电漏斗的残基；ii)鉴定易位途径中的底物结合部位(S)，并评估选定的CTP结构域控制底物进入该途径的能力；Iii)确定在均二聚CTP中形成两个CTP单体之间界面的残基，并利用定点自旋标记和硫醇交联法表征在运输过程中发生的配体诱导的构象变化；以及iv)确定能够生长X射线衍射级CTP晶体的条件，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。该项目与健康有关，关系到CTP在生物能量学中的中心作用。因此，疾病(如糖尿病、癌症)中CTP功能的改变是这些病理特征的异常代谢的一个重要方面。因此，阐明底物通过CTP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.