Molecular Basis for Transmembrane Conduction & Signaling

项目摘要/摘要 本项目旨在确定整体跨膜的机制、结构和结构变化。 控制关键跨膜过程的蛋白质，在可以导致改善治疗方法的水平上 人类疾病。前提是分子结构的改变是发挥作用所必需的 跨膜转运体和门控通道，并由调节功能协调。假说 了解结构变化和功能之间的联系为治疗提供了路线图 为稳定构象状态而产生的有机化合物或Fab片段的干预。一位少校 创新是确定膜蛋白和真核生物原子结构的技术和能力，或 人膜蛋白的分辨率足以指导治疗的发展 一种化合物。主要技术包括X射线衍射、电子冷冻显微镜、传输分析、 电生理学。三个目标聚焦于不同类别的跨膜蛋白。目标1侧重于 阐述了最近发现的一类细胞内通道的机制，这些通道控制着 来自植物或真菌的液泡或动物的内溶酶体内的离子和营养物质。一个目标是在 我们对植物中两孔通道TPC1的原子结构测定，并确定了如何调节 离子的传输通过电压、钙离子和磷酸化来实现。目标朝着以下方向发展 人类TPC1，我们结构中的一种抑制物可以治愈小鼠的埃博拉病毒通过 内溶酶体，以及突变导致溶酶体的另一个细胞内通道人类TRPML 储藏病。目标2试图确定支配次级跨膜的机制 运输商和他们的姐妹运输商。目标首先集中在一种高亲和力的磷酸盐转运体上，在那里我们 获得了高分辨结构，进行了22次突变，并记录了运输性质Vmax和Km，以及 缺失自身磷酸转运蛋白的酵母在血浆中表达突变体对其生长的影响 薄膜。我们还关注了乳糖转运蛋白中的突变体，这些突变体第一次转换了结构 在生物运输周期中的状态之间。目标是了解如何绑定和释放 底物与驱动离子、质子的传输相耦合，以了解这一令人惊讶的机制是否 在第二运输者中很常见。这个目标还涉及一种人类葡萄糖转运蛋白，在这种情况下，我们 展示了毒品线索是如何阻止毒贩的。这种转运蛋白与许多癌症有关。目标3旨在 利用我们的人脑水通道蛋白4的原子结构，了解患者抗体与 自身免疫性视神经脊髓炎，有时是致命性疾病。这将打开一条路，询问我们如何 改变这种相互作用，以获得治疗益处。

Project Summary/Abstract This project seeks to determine the mechanisms, structures, and structure change of integral transmembrane proteins that govern critical transmembrane processes, at the level that can lead to improved therapeutics for human disease. The premise is that alterations in molecular structures are necessary for the function of transmembrane transporters and gated channels, and are coordinated by regulatory functions. The hypothesis is that understanding the linkage between structure change and function provides a roadmap for therapeutic intervention by organic compounds or Fab fragments generated to stabilize conformational states. A major innovation is the technology and ability to determine atomic structures of membrane proteins and eukaryotic, or human membrane proteins at a resolution sufficient to instruct in the development of therapeutic development of compounds. Principal technologies include X-ray diffraction, electron cryomicroscopy, transport assays, electrophysiology. Three aims focus on different classes of transmembrane proteins. Aim 1 focuses on elaborating the mechanisms of a recently discovered class of intracellular channels that govern the release of ions and nutrients from the vacuole in plants or fungi, or the endolysosome in animals. One aim is to build on our atomic structure determination of a two-pore channel TPC1 from plants, and to determine how regulation of ion transport by voltage, by calcium ions, and by phosphorylation is brought about. The aim moves toward human TPC1 where an inhibitor seen in our structure can cure mice of Ebola virus that enters the cell through the endolysosome, and to another intracellular channel human TRPML where mutations cause a lysosomal storage disease. Aim 2 seeks to determine the mechanisms that govern secondary transmembrane transporters and their sister uniporters. The aim focuses first on a high affinity phosphate transporter where we obtained high resolution structure, made 22 mutations and recorded transport properties Vmax and Km, and effects on growth of yeast deleted of its own phosphate transporters, expressing the mutants in the plasma membrane. We also focus on mutants in the lactose transporter that for the first time converted the structure between states in the biological transport cycle. The goal is to understand how the binding and release of substrates is coupled to the transport of a driving ion, protons, and to see if this surprising mechanism is common throughout secondary transporters. This aim also addresses a human glucose transporter where we showed how drug leads block the uniporter. This transporter is relevant to many cancers. Aim 3 aims to leverage our atomic structure of human brain aquaporin 4, to understand the binding by patient antibodies with the autoimmune, sometimes lethal disease neuromyelitis optica. This will open the way to ask how we may alter this interaction to therapeutic benefit.