Computational Studies of Sodium Symporters

描述（由申请人提供）：细胞严格调节分子跨膜时间和空间运动的能力对其生存至关重要。这种运动必须以一种选择性的方式进行，以确保细胞质和其他内部隔室的化学成分不受干扰。为了完成这些任务，细胞膜上布满了转运体和通道，这些转运体和通道通常针对特定的细胞类型或细胞器。本研究的主要目的是利用计算方法研究糖转运体vSGLT的构象变化和功能运作。vSGLT是溶质钠同调转运蛋白家族的细菌成员，负责在人类小肠和肾脏中吸附单糖。vSGLT与一个非常大的转运蛋白超家族有关，称为五螺旋反向重复（5HIR）超家族。对其分子作用的进一步了解有可能有助于治疗与2型糖尿病（T2DM）相关的疾病状态和严重脱水的治疗。在Aim 1中，我们将研究Na+和糖释放到细胞中的偶联。我们假设Na+的退出可以让阻断残基移开，让糖逃逸，就像用钥匙打开一扇门一样。5HIR超家族成员的所有结构都具有这些门，因此阐明这一步骤可以为其他共转运蛋白提供广泛的信息。格拉布实验室的计算将由艾布拉姆森和赖特实验室对突变体vsglt的输运分析辅助。我们在Aim 2中的目标是使用计算药物发现来设计vSGLT和hSGLT2的有效抑制剂。hSGLT2是治疗T2DM的药物靶点，所以我们的努力，加上Wright实验室的筛选，可能会带来新的治疗方法。高亲和力的vSGLT抑制剂将为稳定和结晶未知的vSGLT外向结构提供新的工具。在Aim 3中，我们将使用过渡路径采样与gpu加速动力学相结合来生成面向外和面向内的构象之间的路径集合。这些模拟将在分子细节上揭示允许5HIR超家族在Na+梯度存在下移动底物的机械擒纵机制。这些研究将由艾布拉姆森实验室的实验性SAXS/WAXS和DEER测量指导。最后，hSGLT1通过口服补液疗法在严重脱水的治疗中发挥核心作用，自该疗法问世以来，估计每年可挽救100万至300万人的生命。治疗包括摄入葡萄糖/NaCl溶液。葡萄糖和钠在肠内被hSGLT1通过刷状边界膜吸收，随后沉积在血液中。每运输一摩尔葡萄糖伴随着4-6升的水。我们将确定vSGLT如何以及在哪种状态下允许水渗透，我们将探索不同糖对水渗透的影响。这最后一组计算可能建议改进的解决方案，以帮助几个脱水的病人补充水分。1

DESCRIPTION (provided by applicant): 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 is not disturbed. To carry out these tasks, membranes are studded with 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 sugar transporter vSGLT. vSGLT is the bacterial member of the solute sodium symporter family of transporters responsible for adsorption of simple sugars in the small intestine and kidneys of humans. vSGLT is related to a very large superfamily of transporters called the five helix inverted repeat (5HIR) superfamily. An increased understanding of their molecular workings has the potential to help in treating disease states related to type 2 diabetes mellitus (T2DM) and the treatment of severe dehydration. In Aim 1, we will study the coupling of Na+ and sugar release into the cell. We hypothesize that Na+ exit allows blocking residues to move out of the way and allow sugar to escape, much like opening a gate with a key. All structures of 5HIR superfamily members exhibit these gates, so elucidating this step could be widely informative to other cotransporters. Computations in the Grabe lab will be aided by transport assays on mutant vSGLTs in the Abramson and Wright labs. Our goal in Aim 2 is to use computational drug discovery to design potent inhibitors to vSGLT and hSGLT2. hSGLT2 is a drug target for treating T2DM, so our efforts, coupled with screening in the Wright lab, could lead to new therapies. High-affinity inhibitors to vSGLT would provide a new tool for stabilizing and crystallizing the unknown, outward-facing structure of vSGLT. In Aim 3, we will use transition path sampling coupled with GPU-accelerated dynamics to generate the ensemble of paths between the outward-facing and inward-facing conformations. These simulations will reveal, in molecular detail, the mechanical escapement that allows the 5HIR superfamily to move substrates in the presence of a Na+ gradient. These studies will be guided by experimental SAXS/WAXS and DEER measurements in the Abramson lab. Finally, hSGLT1 plays a central role in the treatment of severe dehydration through the use of Oral Rehydration Therapy, which is estimated to save 1-3 millions lives per year since its inception. Treatment + consists of ingestion of a glucose/NaCl solution. The glucose and Na are absorbed across the brush border membrane by hSGLT1 in the intestine and subsequently deposited in the blood. Each transported mole of glucose is accompanied by 4-6 L of water. We will determine how and in which state(s) vSGLT allows water to permeate, and we will explore the effect of different sugars on water permeation. This final set of computations may suggest improved solutions to aid in rehydration of severally dehydrated patients. 1