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.

抽象的 膜转运蛋白的氨基酸-多胺-有机阳离子 (APC) 超家族是其中之一 最大的溶质载体家族。它们参与各个领域的营养和氨基酸运输 生活。在人类中，它们对于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 是多种多样的，它们采用了一种称为“LeuT折叠”的共同拓扑排列，涉及倒置的 5 + 5 个跨膜螺旋的拓扑重复，在 C 末端还有 2 个以上的螺旋参与 某些异构体中的寡聚化。近年来，许多LeuT折叠同源物的结构已被解析 以及运输周期中几乎每个州。在检查这些结构时，存在一个主要问题 out - 为什么 LeuT 折叠转运蛋白以不同的寡聚形式（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 转运蛋白中发挥作用 调节，并且所有 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 保守的亚基结构。然而，由于不同的稳定性，观察到的低聚物存在多样性 由于蛋白质和脂质接触表面的差异。为了研究这个问题，我们将研究一对 APC 运输商为严格调查这个问题提供了一个模型系统。这些是 精氨酸/胍丁胺逆向转运蛋白 AdiC 和谷氨酸/GABA 逆向转运蛋白 GadC。这两个运输机是 在同一亚科中具有进化相关性，并且都参与大肠杆菌的极端耐酸性，但是， AdiC 仅作为二聚体被观察到，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来研究 LeuT 折叠氨基酸转运蛋白的二聚化决定因素。在本次调查中，我们将使用组合 跨学科研究使我们能够检查其他膜蛋白的二聚稳定性 系统，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 绘制出蛋白质伙伴、与水或脂质的相互作用以及相关和解离的膜结构 形式。将进行单分子光漂白分析实验来测量平衡 膜中亚基的二聚化亲和力，同时测试膜中确定的关键蛋白质和脂质变量 计算分析。在目标 1 中，我们将找出 AdiC 二聚化的定义，在目标 2 中，确定 定义使 GadC 成为单体的特征，并通过最终测试将 GadC 设计为功能性二聚体。这 两种模型 LeuT 折叠转运蛋白之间的严格比较/对比研究将为 了解与所有 APC 转运蛋白相关的寡聚机制，包括那些重要的机制 在人类健康和疾病中。