Determinants of amino acid transporter oligomerization in membranes

膜中氨基酸转运蛋白寡聚的决定因素

• 批准号: 10725968
• 负责人: Janice L Robertson
• 金额: $ 15.55万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725968
• 关键词: Acids; Address; Adopted; Affinity; Agmatine; Amino Acid Transporter; Amino Acids; Arginine; Binding; Biological Assay; Biological Models; Blood - brain barrier anatomy; CLC Gene; Cell physiology; Computer Analysis; Couples; Coupling; Dimerization; Disease; Dissociation; Engineering; Equilibrium; Escherichia coli; Essential Amino Acids; Family; Fluorescence Resonance Energy Transfer; Free Energy; Glutamates; Grain; Health; Homologous Gene; Homology Modeling; Human; Interdisciplinary Study; Investigation; Ion Channel; Kidney; Kinetics; Label; Life; Lipids; Maps; Measures; Membrane; Membrane Proteins; Membrane Transport Proteins; Microscopy; Modeling; Molecular; Molecular Conformation; Movement; Nutrient; Photobleaching; Physiological; Polyamines; Protein Isoforms; Proteins; Reaction; Regulation; Reporting; Research; Resistance; Role; Sampling; Site; Structure; Surface; Synapses; System; Testing; Variant; Water; antiporter; computer studies; dimer; experimental analysis; experimental study; fluorophore; gamma-Aminobutyric Acid; innovation; interdisciplinary approach; membrane assembly; molecular dynamics; molecular modeling; monomer; neurophysiology; neurotransmitter reuptake; reconstitution; single molecule; solute

ABSTRACT The Amino Acid-Polyamine-organoCation (APC) superfamily of membrane transport proteins is one of the largest families of solute carriers. They are involved in nutrient and amino acid transport across all kingdoms of life. In humans, they are essential for amino acid transport in kidneys, neurotransmitter re-uptake in synapses, movement of substances across the blood-brain barrier, and many other physiological roles. While their functions are diverse, they adopt a common topological arrangement known as the "LeuT-fold", involving an inverted topology repeat of 5 + 5 transmembrane helices, with an additional 2+ helices at the C-terminus that engage in oligomerization in some isoforms. In recent years, structures of many LeuT-fold homologues have been solved and in nearly every state along the transport cycle. When examining these structures, a major question stands out - why are LeuT-fold transporters observed in diverse oligomeric forms (i.e. monomers, dimers and trimers) despite possessing the same subunit fold? We hypothesize that oligomerization plays a role in APC transporter regulation, and that all LeuT-fold transporters possess an innate potential to dimerize in membranes due to the conserved subunit structure. However, the diversity of observed oligomers arises because of different stabilities due to differences in protein and lipid contact surfaces. To investigate this, we will study a pair of APC transporters that provide a model system for the rigorous investigation of this question. These are the arginine/agmatine antiporter AdiC and glutamate/GABA antiporter GadC. These two transporters are evolutionarily related within the same sub-family, and are both involved in extreme acid resistance in E. coli, yet, AdiC has only ever been observed as a dimer, while GadC is monomeric. In addition, these proteins are easy to purify and can be studied functionally in reconstituted systems, providing rigorous model systems to study dimerization determinants of LeuT-fold amino-acid transporters. In this investigation, we will use a combination of interdisciplinary studies that have allowed us to examine dimerization stability of other membrane protein systems, such as the CLC Cl-/H+ antiporter and the dual-topology Fluc F- ion channel. Computational studies will map out protein partners, interactions with water or lipids, and membrane structure in associated and dissociated forms. Single-molecule photobleaching analysis experiments will be carried out to measure the equilibrium dimerization affinity of subunits in membranes while testing key protein and lipid variables identified in the computational analysis. In Aim 1, we will map out what defines AdiC dimerization, and in Aim 2, identify the defining features that make GadC monomeric, with a final test to engineer GadC as a functional dimer. This rigorous compare/contrast study between two model LeuT-fold transporters will provide a fundamental understanding of oligomerization mechanisms relevant to all APC transporters, including those that are important in human health and disease.

摘要 膜转运蛋白的氨基酸-多胺-有机阳离子（APC）超家族是膜转运蛋白家族中的一个。 最大的溶质载体家族。它们参与营养和氨基酸在所有生物界的运输 生活在人类中，它们对肾脏中的氨基酸运输、突触中的神经递质再摄取， 物质穿过血脑屏障的运动，以及许多其他生理作用。虽然它们的功能 是不同的，它们采用一种共同的拓扑结构，称为“LeuT折叠”，涉及一个倒置的 5 + 5个跨膜螺旋的拓扑重复，在C-末端有另外的2+个螺旋， 在一些同种型中的寡聚化。近年来，许多LeuT折叠同源物的结构已被解决 并且几乎在沿着运输周期的沿着每个状态中。在研究这些结构时， 为什么LeuT折叠转运蛋白以不同的寡聚体形式（即单体，二聚体和三聚体）被观察到 尽管拥有相同的亚基折叠我们假设寡聚化在APC转运蛋白中起作用， 所有LeuT-折叠转运蛋白都具有在膜中二聚化的先天潜力，这是由于 保守亚基结构然而，由于不同的稳定性，观察到的寡聚体的多样性增加 这是由于蛋白质和脂质接触表面的差异。为了研究这一点，我们将研究一对APC 运输商，提供了一个模型系统，为这个问题的严格调查。这些都是 精氨酸/胍丁胺反向转运体AdiC和谷氨酸/GABA反向转运体GadC。这两个运输机是 在进化上属于同一个亚家族，并且都与E.大肠杆菌，然而， AdiC仅被观察到为二聚体，而GadC是单体。此外，这些蛋白质很容易 纯化，并可以在重构系统中进行功能研究，提供严格的模型系统进行研究 LeuT折叠氨基酸转运蛋白的二聚化决定簇。在这次调查中，我们将结合 跨学科研究使我们能够检查其他膜蛋白的二聚化稳定性 例如CLC Cl-/H+反向转运蛋白和双拓扑Fluc F-离子通道。计算研究将 绘制蛋白质伴侣，与水或脂质的相互作用，以及相关和解离的膜结构 forms.单分子光漂白分析实验将进行测量的平衡 在膜中亚基的二聚化亲和力，同时测试在细胞膜中鉴定的关键蛋白和脂质变量。 计算分析在目标1中，我们将绘制出定义AdiC二聚化的内容，并在目标2中，确定AdiC二聚化的定义。 定义使GadC单体的特征，最终测试将GadC工程化为功能性二聚体。这 两个模型LeuT折叠转运蛋白之间的严格比较/对比研究将提供基本的 理解与所有APC转运蛋白相关的寡聚化机制，包括那些重要的 人类健康和疾病。