Dynamics and catalysis in integral membrane pyrophosphatases

整合膜焦磷酸酶的动力学和催化

• 批准号: BB/T006048/2
• 负责人: Christos Pliotas
• 金额: $ 21万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT006048%2F2
• 关键词: Dynamics; catalysis; integral; membrane; pyrophosphatases

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.

60%的药物靶标是完整的膜蛋白，但只占所有已解决结构的3%。此外，对膜蛋白的快速动力学分析仅限于细胞色素c氧化酶等蛋白质。整体膜焦磷酸酶(MPPase)是一种进化保守的离子泵，它能将焦磷酸盐中的自由能转化为跨膜的钠和/或质子梯度。它们与任何其他蛋白质不同，不存在于多细胞动物中，在低能量应激条件下是必不可少的。除了植物和(古)细菌外，mPPase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(Nagana，昏睡病)、弓形虫(感染高达90%的猪)和疟原虫(疟疾)，以及最常见的脑脓肿原因--普通拟杆菌(死亡率为20%)。这些疾病在世界大部分地区影响人类健康和粮食安全，除疟疾外的原生动物疾病被归类为“被忽视的热带疾病”。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年的北欧夏季常见。我们已经证明，在恶性疟原虫中删除mPPase基因使其具有非传染性。因此，mPPase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。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新的武器，从而对动物健康、食品安全和人类疾病具有重要的长期好处。本工作扩展和深化了细菌Na+泵Thermotoga maritima mPPase(TmPPase)和H+泵Vigna Radiata(绿豆)mPPase(VrPPase)的开创性结构。在BBSRC之前的资助下，我们开发了四种新型的mPPase抑制剂支架，其中三种在低Um浓度下对疟疾寄生虫具有活性。这些分子以意想不到的方式工作，以变构的方式阻断出口通道。我们的愿景是扩大我们的结构研究，使用单分子功能、时间分辨结晶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)专注于了解不同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，对钠(Na)和钾(K)浓度的变化做出不同的反应；以及(2)使用各种动态方法(单分子荧光共振能量转移(FRET)、时间分辨序列同步加速器结晶学(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。这两方面的工作相互影响，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从而离子泵浦，钠泵和质子泵浦mPPase之间的这些变化有何不同，以及结合和泵浦构象变化是如何在两个单体之间变构传递的，从而导致半位置的反应性。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关于这种酶的详细程度方面是革命性的。