Computational studies of sodium symporters

钠同向转运体的计算研究

• 批准号: 9311724
• 负责人: Michael Grabe
• 金额: $ 37.04万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9311724
• 关键词: Address; Adsorption; Affinity; Amino Acid Neurotransmitters; Binding; Binding Proteins; Binding Sites; Biological Assay; Cells; Chemicals; Chemistry; Collaborations; Collection; Computing Methodologies; Coupled; Coupling; Crystallization; Cytoplasm; Data; Dehydration; Disease; Drug Design; Electrophysiology (science); Energy Metabolism; Ensure; Environment; Event; FDA approved; Face; Family; Goals; Grant; Homologous Gene; Homology Modeling; Human; Ions; Kidney; Kinetics; Lead; Leucine; Ligands; Light; Liposomes; Measurement; Membrane; Mental Depression; Metabolism; Methods; Modeling; Molecular; Molecular Conformation; Movement; Mutate; Non-Insulin-Dependent Diabetes Mellitus; Organelles; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Play; Proteins; Pump; Resolution; Role; Sampling; Sialic Acids; Site; Small Intestines; Sodium; Sodium Iodide; Structure; Testing; Thyroid Gland; Time; Transmembrane Transport; Validation; Water; base; biophysical tools; cell type; computer studies; conformational conversion; design; experimental study; extracellular; improved; inhibitor/antagonist; member; molecular dynamics; next generation; novel; operation; prospective; reconstitution; screening; serotonin transporter; sialic acid permease; simulation; small molecule; solute; spectroscopic data; sugar; symporter; tool; uptake; virtual

Project Summary/Abstract The ability of the cell to tightly regulate the temporal and spatial movement of molecules across membranes is central to its survival. This movement has to be done in a selective manner to ensure that the chemistry of the cytoplasm and other internal compartments are not disturbed. To carry out these tasks, membranes contain transporters and channels that are often specific to particular cell types or organelles. The primary objective of the current proposal is to use computational methods to examine the conformational changes and functional operation of the SGLT sugar cotransporters and the closely related sialic acid transporter nanT. Most of our efforts are focused on vSGLT, the bacterial member of the solute sodium symporter family of cotransporters, whose human homologues are responsible for adsorption of simple sugars in the small intestine and kidneys. vSGLT is related to a very large superfamily of transporters called the Leucine Transporter (LeuT) superfamily, which include serotonin transporters, sodium iodide transporters and other important pharmacological targets. An increased understanding of the molecular workings of these transporters has the potential to help in treating diseases related to type 2 diabetes mellitus (T2DM), severe dehydration, and depression. In Aim 1, we will study how Na+ and substrate bind to the outward-facing state of cotransporters, an important first step in recognition and entry into the cell. We hypothesize that Na+ binds first to prime the protein for substrate binding. We will take advantage of our collaborator's recent determination of the high-resolution structure of the sialic acid transporter (nanT) at 2.0 Å in the outward-facing state. We will then compute the energetics of outer gate closing. We posit that cargo loading will help stabilize the gate in a closed conformation. Next, we will create an outward-facing model of vSGLT based on nanT and validate the model with experiments in the Abramson lab (UCLA) such as DEER distance measurements (with Mchaourab lab, Vanderbilt), WAXS studies (with Neutze lab, Gothenburg), and uptake assays. Extracellular sugar and Na+ binding will then be studied using computation. Our goal in Aim 2 is to use computational drug design to reveal the structural basis of inhibitor binding to human SGLT2 (a T2DM target) and find small molecules that bind vSGLT in a conformationally selective manner. Our efforts on hSGLT2 will be coupled with screening in the Wright lab (UCLA), which could lead to improved T2DM therapies. Meanwhile, small molecules that bind vSGLT in distinct states, which do not exist, would provide tools for stabilizing and crystallizing the unknown, outward-facing structure of vSGLT as well as interpreting spectroscopic data. In Aim 3, we will use enhanced sampling methods (such as the Weighted Ensemble method or Markov State Modeling) to simulate the entire transport cycle and reveal how Na+ and substrate drive the cotransporter through key conformational states – thus revealing the mechanistic underpinnings of membrane transport in this important superfamily.

项目总结/摘要 细胞严格调节分子跨膜的时空运动的能力， 对它的生存至关重要这种运动必须以选择性的方式进行，以确保 细胞质和其他内部隔室不受干扰。为了完成这些任务，膜包含 转运蛋白和通道，往往是特定的细胞类型或细胞器。的主要目的 目前的建议是使用计算方法来检查构象变化和功能 SGLT糖协同转运蛋白和密切相关的唾液酸转运蛋白nanT的运作。我们的大多数 努力集中在vSGLT，共转运蛋白的溶质钠同向转运蛋白家族的细菌成员， 其人类同系物负责小肠和肾脏中单糖的吸收。 vSGLT与称为亮氨酸转运蛋白（LeuT）超家族的非常大的转运蛋白超家族相关， 包括5-羟色胺转运蛋白、碘化钠转运蛋白和其它重要的药理学靶点。 对这些转运蛋白的分子工作机制的进一步了解有可能有助于治疗 与2型糖尿病（T2 DM）、严重脱水和抑郁症相关的疾病。 在目标1中，我们将研究Na+和底物如何与共转运蛋白的外向状态结合，这是一个重要的 识别和进入细胞的第一步。我们假设Na+首先结合蛋白质， 底物结合我们将利用我们的合作者最近确定的高分辨率 唾液酸转运蛋白（nanT）的结构在2.0 ℃下朝外状态。然后我们将计算 关闭外门的能量学。我们认为货物装载将有助于稳定大门， 构象接下来，我们将基于nanT创建一个面向外部的vSGLT模型，并验证该模型 利用Abramson实验室（UCLA）中的实验，例如DEER距离测量（与Mchaourab实验室， 范德比尔特）、WAXS研究（与Neutze实验室，哥德堡）和摄取测定。胞外糖和Na+ 然后将使用计算来研究绑定。我们的目标2是使用计算药物设计来揭示 抑制剂与人SGLT 2（一种T2 DM靶点）结合的结构基础，并发现结合的小分子 vSGLT在构象选择性的方式。我们在hSGLT 2上的努力将与筛选相结合， 赖特实验室（加州大学洛杉矶分校），这可能导致改善T2 DM治疗。与此同时， 处于不同状态的vSGLT，并不存在，将提供稳定和明确未知的工具， vSGLT的外向结构以及解释光谱数据。在目标3中，我们将使用增强的 采样方法（如加权包络法或马尔可夫状态建模）来模拟整个 运输周期，并揭示Na+和底物如何通过关键构象状态驱动协同转运蛋白- 从而揭示了这一重要超家族中膜转运的机制基础。