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Structure/Function of Mitochondrial Citrate Carrier

线粒体柠檬酸盐载体的结构/功能

基本信息

批准号：
7932586
负责人：
Ronald Sloan Kaplan
金额：
$ 13.05万
依托单位：
ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7932586
关键词：
Amino Acids Antibodies Award Binding Binding Sites Bioenergetics Carrier Proteins Charge Chemicals Citrates Classification Coupling Crystallization Crystallography Cysteine Detergents Diabetes Mellitus Disease Docking Electrostatics Energy Metabolism Engineering Eukaryotic Cell Fab Immunoglobulins Foundations Generations Growth Health Homology Modeling Individual Inner mitochondrial membrane Kinetics Laboratories Letters Ligands Lipids Location Malignant Neoplasms Manuals Maps Measures Membrane Membrane Proteins Metabolism Methodology Methods Mitochondria Modeling Modification Molecular Molecular Conformation Molecular Structure Monoclonal Antibodies Movement Mutagenesis Mutate Mutation Nature Pathology Pathway interactions Phase Phosphoenolpyruvate Play Positioning Attribute Preparation Procedures Production Property Proteins Protocols documentation Reagent Relative (related person)Research Personnel Resolution Roentgen Rays Role Scanning Screening procedure Series Side Site Solvents Spin Labels Structure Structure-Activity Relationship Substrate Specificity Sulfhydryl Compounds Surface Tertiary Protein Structure Testing Transmembrane Domain Transport Reaction Water X ray diffraction analysis X-Ray Diffraction antiporter base citrate carrier crosslink defined contribution design dicarboxylate dimer flexibility inhibitor/antagonist insight interest methanethiosulfonate molecular dynamics monomer mutant programs protein function protein structure research study tricarboxylate

项目摘要

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.

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）的分子结构及其转运机制之间的关系。该转运蛋白催化三羧酸盐、二羧酸盐和磷酸烯醇丙酮酸穿过线粒体内膜的交换，因此对于真核细胞的能量代谢至关重要。最近，我们： i) 对跨膜结构域 (TMD) HI 和 IV 进行半胱氨酸扫描诱变研究，结合化学修饰、硝基氧扫描和底物保护实验，可以鉴定底物易位途径的重要部分； ii) 开发了CTP结构的同源模型； iii) 开发了在结晶相容洗涤剂中纯化 CTP 的方法，从而启动了全面的结晶试验。在此基础上，我们建议开展研究，以继续从根本上推进我们对这种代谢重要转运蛋白功能的理解。具体来说，将进行实验：i) 定义其余四个 TMD 在 CTP 底物易位途径形成中的贡献（通过在我们的同源模型 CTP 结构的基础上选择的位置进行半胱氨酸取代诱变，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，该漏斗将柠檬酸盐从其表面吸引到途径中； ii) 识别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 中两个 CTP 单体之间形成界面的残基，并使用定点自旋标记和硫醇交联表征运输过程中发生的配体诱导的构象变化； iv) 确定能够生长 X 射线衍射质量 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。该项目的健康相关性涉及 CTP 在生物能学中的核心作用。因此，疾病（例如糖尿病、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 进行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。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins.

野生型和无半胱氨酸酵母线粒体柠檬酸转运蛋白的寡聚状态。

DOI：
10.1023/a:1005460810527
发表时间：
1999
期刊：
Journal of bioenergetics and biomembranes
影响因子：
3
作者：
Kotaria,R;Mayor,JA;Walters,DE;Kaplan,RS
通讯作者：
Kaplan,RS

The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.

酵母线粒体柠檬酸转运蛋白：使用定点自旋标记确定跨膜结构域 IV 的二级结构和溶剂可及性。

DOI：
10.1021/bi000433e
发表时间：
2000
期刊：
Biochemistry
影响因子：
2.9
作者：
Kaplan,RS;Mayor,JA;Kotaria,R;Walters,DE;McHaourab,HS
通讯作者：
McHaourab,HS

Mitochondrial and Plasma Membrane Citrate Transporters: Discovery of Selective Inhibitors and Application to Structure/Function Analysis.

DOI：
发表时间：
2010-06
期刊：
Molecular and cellular pharmacology
影响因子：
0
作者：
Jiakang Sun;Sreevidya Aluvila;R. Kotaria;J. Mayor;D. Walters;R. S. Kaplan
通讯作者：
Jiakang Sun;Sreevidya Aluvila;R. Kotaria;J. Mayor;D. Walters;R. S. Kaplan

The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate.

酵母线粒体柠檬酸转运蛋白：鉴定负责吡哆醛 5-磷酸介导的抑制作用的赖氨酸残基。

DOI：
10.1007/s10863-008-9187-1
发表时间：
2008
期刊：
Journal of bioenergetics and biomembranes
影响因子：
3
作者：
Remani,Sreevidya;Sun,Jiakang;Kotaria,Rusudan;Mayor,JuneA;Brownlee,JuneM;Harrison,DavidHT;Walters,DEric;Kaplan,RonaldS
通讯作者：
Kaplan,RonaldS

Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach.

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
发表时间：
2010
期刊：
Journal of bioenergetics and biomembranes
影响因子：
3
作者：
Mayor,JuneA;Sun,Jiakang;Kotaria,Rusudan;Walters,DEric;Oh,KyoungJoon;Kaplan,RonaldS
通讯作者：
Kaplan,RonaldS