Computational studies of sodium symporters

项目摘要/摘要 细胞严密调节分子跨膜的时空运动的能力是 对它的生存至关重要。这一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。为了执行这些任务，膜包含 通常是特定细胞类型或细胞器所特有的转运体和通道。的主要目标是 目前的建议是使用计算方法来研究构象变化和功能 SGLT糖转运体和密切相关的唾液酸转运体NANT的运行。我们的大多数人 研究的重点是vSGLT，它是共转运体中溶质钠转运体家族的细菌成员， 它们的人类同系物负责在小肠和肾脏中吸收单糖。 VSGLT与一个非常大的转运蛋白超家族相关，称为亮氨酸转运蛋白(Leut)超家族， 其中包括5-羟色胺转运体、碘化钠转运体和其他重要的药理靶点。 对这些转运蛋白分子工作原理的进一步了解有可能有助于治疗 与2型糖尿病(T2 DM)、严重脱水和抑郁有关的疾病。 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+首先与蛋白质结合，为 底物结合。我们将利用我们的合作者最近对高分辨率 唾液酸转运体(NANT)的结构，在向外的状态下，在2.0ä。然后我们将计算 关闭外门的能量学。我们假定货物装载将有助于在关闭的状态下稳定闸门. 构象。接下来，我们将基于NANT创建vSGLT的面向外部的模型，并对该模型进行验证 利用艾布拉姆森实验室(UCLA)的实验，例如鹿的距离测量(利用Mchaourab实验室， WAXS研究(与哥德堡的Neutze实验室)和摄取分析。胞外糖与钠离子 然后将使用计算来研究结合。我们在目标2中的目标是使用计算机药物设计来揭示 抑制物与人SGLT2(T2 DM靶标)结合的结构基础及寻找结合的小分子 VSGLT以构象选择性的方式表达。我们在hSGLT2方面的努力将与在 莱特实验室(UCLA)，这可能会导致T2 DM治疗的改进。与此同时，结合在一起的小分子 处于不同状态的vSGLT将为稳定和结晶未知提供工具， VSGLT的外向结构以及对光谱数据的解释。在目标3中，我们将使用增强版 抽样方法(如加权系列法或马尔可夫状态建模)来模拟整个 运输循环并揭示Na+和底物如何通过关键构象状态驱动共转运蛋白- 从而揭示了这个重要的超家族中膜运输的机械基础。

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.