Dynamics and catalysis in integral membrane pyrophosphatases

60%的药物靶点是完整的膜蛋白，但在所有已解决的结构中仅占3%。此外，膜蛋白的快速动力学分析仅限于细胞色素c氧化酶等蛋白。整体膜焦磷酸酶（mPPases）是进化上保守的离子泵，它将焦磷酸盐中的自由能转化为跨膜的钠和/或质子梯度。它们不同于任何其他蛋白质，不会出现在多细胞动物中，并且在低能量应激条件下是必不可少的。除了植物和（古）细菌外，mpp酶还出现在病原体中：原生动物寄生虫，如利什曼原虫（利什曼病）、锥虫（昏睡病）、刚地弓形虫（感染高达90%的猪）和疟原虫（疟疾），以及普通拟杆菌，后者是导致脑脓肿的最常见原因（死亡率为20%）。这些疾病影响着世界大部分地区的人类健康和粮食安全，除疟疾外，原生动物疾病属于“被忽视的热带病”。由于全球变暖，传播这些疾病的昆虫媒介已经蔓延到欧洲，并将在未来30年内在北欧的夏季普遍存在。我们已经证明，删除恶性疟原虫的mPPase基因使其不具有传染性。因此，mPPases是一个潜在的药物靶点，我们的初步工作表明它适合进行动力学分析。通过提供对抗主要动物和人类疾病的新武器，开发针对这些酶的药物将对动物健康、食品安全和人类疾病具有重要的长期利益。这项工作扩展和深化了我们突破性的细菌Na+泵送Thermotoga martima mPPase （TmPPase）和H+泵送Vigna radiata（绿豆）mPPase （VrPPase）结构。在之前的BBSRC资助下，我们开发了四种新型mPPase抑制剂支架，其中三种在低uM浓度下对疟原虫具有活性。这些分子以意想不到的方式起作用，以变构的方式阻断出口通道。我们的愿景是扩展我们的结构研究，并使用单分子功能，时间分辨晶体学和分子动力学模拟来确定中间酶状态。我们的多学科方法有两个主要方面：(1)专注于理解不同mPPases背后的结构相关性。至少有五个不同的家族，它们泵送不同的离子，对钠（Na）和钾(K)浓度的变化有不同的反应；(2)利用各种动态(单分子荧光共振能量转移（FRET），时间分辨串行同步加速器晶体学（SSX）和溶液（脉冲电子-电子双共振（PELDOR））方法来了解酶的机制。这两方面的工作相互联系，因为静态结构研究将产生可以通过生物物理技术测试的假设。我们的目的是了解螺旋中导致门打开和离子泵送的运动，这些运动在钠泵送和质子泵送mppase之间有何不同，以及结合和泵送构象变化如何在两个单体之间变构传递，从而导致半位反应性。这项工作将使用我们开发的新的变构抑制剂。我们期望我们的工作在我们获得的关于这种酶的细节水平上是革命性的。

60% of drug targets are integral membrane proteins - but just 3% of all solved structures. In addition, fast kinetic analysis on membrane proteins has been restricted to proteins like cytochrome c oxidase. Integral membrane pyrophosphatases (mPPases) are evolutionarily conserved ionic pumps that convert the free energy in pyrophosphate into a sodium and/or proton gradient across a membrane. They are unlike any other protein, do not occur in multicellular animals, and are essential under conditions of low-energy stress. In addition to plants and (archae)bacteria, mPPases occur in pathogens: protozoan parasites like Leishmania (leishmaniasis), Trypanosoma species (Nagana, sleeping sickness), Toxoplasma gondii (infecting up to 90% of pigs) and Plasmodium species (malaria), as well as Bacteroides vulgatus, which is the most common cause of brain abscesses (20% mortality rate). These diseases affect human health and food security across much of the world, and the protozoan diseases, except for malaria, are classes as "neglected tropical diseases". Due to global warming, the insect vectors that spread these diseases are already spreading into Europe and will be common in the summer in Northern Europe in the next 30 years. We have shown that deleting the mPPase gene in P. falciparum makes it non-infectious. mPPases are thus a potential drug target, and our preliminary work suggests it is suitable for kinetic analysis. Developing drugs against these enzymes will have important long-term benefits for animal health, food security, and human disease, by providing new weapons against major animal and human diseases.This work extends and deepens our ground-breaking structures of the bacterial Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata (mung bean) mPPase (VrPPase). With previous BBSRC funding, we developed four novel mPPase inhibitor scaffolds, three of which are active against the malaria parasite at low uM concentrations. The molecules work in unexpected ways, by blocking the exit channel in an allosteric manner. Our vision is to extend our structural studies and use single molecule functional, time-resolved crystallography and molecular dynamics simulations to determine intermediate enzymatic states. Our multidisciplinary approach has two main strands: (1) focussing on understanding the structural correlates behind the different mPPases. There are at least five different families, which pump different ions and respond differently to changes in sodium (Na) and potassium (K) concentration; and (2) using various dynamic (single-molecule fluorescence resonance energy transfer (FRET), time-resolved serial synchrotron crystallography (SSX) and solution (Pulsed Electron-Electron Double Resonance (PELDOR)) approaches to understand the choreography of the enzyme mechanism. The two strands of work inform each other, as the static structural studies will generate hypotheses that can be tested by biophysical techniques.Our aim is to understand what motions in the helices leading to gate opening and thus ion pumping, how these differ between sodium- and proton-pumping mPPases, and how the binding and pumping conformational changes are allosterically transmitted between the two monomers, leading to half-of-the-sites reactivity. The work will use the new allosteric inhibitors that we have developed. We expect our work to be revolutionary in the level of detail we obtain about this enzyme.