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 - 聚氨酰胺 - 有机（APC）是其中之一 最大的溶质承运人家族。他们参与了跨越所有王国的营养和氨基酸运输 生活。在人类中，它们对于在肾脏中的氨基酸转运至关重要，神经递质在突触中重新摄取， 物质在血脑屏障中的运动以及许多其他生理作用。而他们的功能 有多样化，他们采用一种称为“ leut折叠”的共同拓扑布置，涉及倒置 拓扑重复5 + 5跨膜螺旋，在C末端有2+螺旋的螺旋螺旋 在某些同工型中的寡聚。近年来，已经解决了许多Leut-fold同源物的结构 在运输周期中几乎每个州。在检查这些结构时，一个主要的问题是 出局 - 为什么在不同的寡聚形式（即单体，二聚体和三聚体）中观察到Leut折叠转运蛋白 尽管拥有相同的亚基折？我们假设低聚在APC转运蛋白中起作用 调节，所有Leut折叠转运蛋白具有固有的潜力，可以在膜上二聚 保守的亚基结构。但是，观察到的低聚物的多样性是由于不同的稳定性而产生的 由于蛋白质和脂质接触表面的差异。为了调查这一点，我们将研究一对APC 为对该问题进行严格研究的模型系统的运输商。这些是 精氨酸/氨木他的抗植物ADIC和谷氨酸/GABA抗植物GADC。这两个转运蛋白是 在同一子家庭中的进化相关，并且都参与大肠杆菌中的极高酸性，但是 ADIC仅被视为二聚体，而GADC是单体。另外，这些蛋白质很容易 净化并可以在重构系统中进行功能研究，并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。在这项调查中，我们将使用一个组合 跨学科研究使我们能够检查其他膜蛋白的二聚化稳定性 系统，例如CLC CL-/H+抗胞植物和双倍型FLUC F-ION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 表格。将进行单分子光漂白分析实验以测量平衡 在测试关键蛋白和脂质变量时，亚基在膜上的二聚化亲和力 计算分析。在AIM 1中，我们将绘制出定义ADIC二聚体化的内容，并在AIM 2中确定 定义使GADC单体的特征，并对工程师GADC作为功能二聚体进行了最终测试。这 严格的比较/对比研究，两个模型leut折叠转运蛋白之间将提供基本 了解与所有APC转运蛋白有关的寡聚机制，包括重要的转运蛋白 在人类健康和疾病中。