Dynamics and catalysis in integral membrane pyrophosphatases

60%的药物靶点是完整的膜蛋白-但只有3%的所有解决的结构。此外，对膜蛋白的快速动力学分析已被限制为蛋白质，如细胞色素c氧化酶。整合膜焦磷酸酶（mPPase）是进化上保守的离子泵，其将焦磷酸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，不存在于多细胞动物中，并且在低能量应激条件下是必需的。除了植物和（古）细菌外，mPP酶还存在于病原体中：原生动物寄生虫，如利什曼原虫（利什曼病），锥虫属（Nagana，昏睡病），弓形虫（感染高达90%的猪）和疟原虫属（疟疾），以及普通拟杆菌，这是脑水肿的最常见原因（20%死亡率）。这些疾病影响到世界许多地区的人类健康和粮食安全，而除疟疾外，原生动物疾病被列为“被忽视的热带疾病”。由于全球变暖，传播这些疾病的昆虫病媒已经在向欧洲蔓延，未来30年将在北方欧洲的夏季常见。我们已经证明，删除恶性疟原虫中的mPPase基因使其无感染性。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于动力学分析。开发针对这些酶的药物将为动物健康、食品安全和人类疾病提供重要的长期利益，为主要动物和人类疾病提供新的武器。这项工作扩展和深化了我们对细菌Na+泵海栖热袍菌mPPase（TmPPase）和H+泵绿豆mPPase（VrPPase）的突破性结构。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浓度下对疟疾寄生虫有活性。这些分子以意想不到的方式工作，通过以变构方式阻断出口通道。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的状态。我们的多学科方法有两个主要方面：（1）专注于理解不同mPPase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并对钠（Na）和钾（K）浓度的变化做出不同的响应;以及（2）使用各种动态（单分子荧光共振能量转移（FRET）、时间分辨连续同步晶体学（SSX）和溶液（脉冲电子-电子双共振（PELDOR））方法来理解酶机制的编排。这两条工作链相互通报，因为静态结构研究将产生可以通过生物物理技术检验的假设。我们的目标是了解螺旋中的哪些运动导致门打开并因此导致离子泵送，这些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，导致一半的位点反应性。这项工作将使用我们开发的新的变构抑制剂。我们希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.