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％的药物靶标是整体膜蛋白，但仅占所有解决结构的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）是进化保守的离子泵，可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，在多细胞动物中不会发生，并且在低能应激的条件下至关重要。除植物和（弓形）细菌外，MPPase还发生在病原体中：诸如利什曼原虫（利什曼病），锥虫瘤物种，锥虫症（长子，睡眠疾病），弓形虫弓形虫（弓形虫（Toxoplasma gondii （20％的死亡率）。这些疾病会影响世界上许多地方的人类健康和粮食安全，除疟疾外，原生动物疾病是“被忽视的热带疾病”的类。由于全球变暖，传播这些疾病的昆虫向量已经扩散到欧洲，并且在未来30年的夏季将很普遍。我们已经表明，在恶性疟原虫中删除MPPase基因使其不感染。因此，MPPase是一个潜在的药物靶标，我们的初步工作表明它适合动力学分析。针对这些酶开发药物将通过提供针对主要动物和人类疾病的新武器来对动物健康，粮食安全和人类疾病具有重要的长期益处。这项工作扩展并加深了我们对细菌Na+pumpumpumping Thermotoga Maritima maritima maritima mppase（TMPPase）Mppase（TMPPase）（TMPPASE）（TMPPSE）和H+-Pumpumpumping Vigna radiata radiata radiata radiata radiata（VR）（MM）的突破性结构。通过以前的BBSRC资助，我们开发了四个新型的MPPase抑制剂支架，其中三个在低浓度下对疟疾寄生虫具有活性。该分子以意外的方式工作，通过以变构的方式阻止出口通道。我们的视野是扩展我们的结构研究，并使用单分子功能，时间分辨的晶体学和分子动力学模拟来确定中间酶态。我们的多学科方法有两个主要链：（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，它们会泵送不同的离子，并且对钠（Na）和钾（K）浓度的变化的反应不同。和（2）使用各种动态（单分子荧光共振能量转移（FRET），时间分辨的串行同步晶体学（SSX）（SSX）（SSX）和溶液（脉冲电子双共振（PELDOR））方法，以了解酶促机制的综合性均匀的研究。生物物理技术的目的是了解导致门开口的螺旋中的动作，从而在钠和质子泵的mppase之间有所不同，以及结合和泵构的变化如何在两个单体之间变式地传播，从而导致了一半的旋转。关于这种酶。