Exploiting the structure of integral membrane pyrophosphatases

尽管它们是目前60%的药物的靶标，但在蛋白质数据库中，整体膜蛋白只占不到2%的结构。此外，对它们的快速动力学研究大多局限于具有发色团的分子，如细胞色素c氧化酶。这项工作将建立在我们2012年发表的开创性的海洋热藻Na+泵送焦磷酸酶（TmPPase） x射线结构的基础上。整体膜焦磷酸酶（mPPases）是进化上保守的新型“初级”离子泵，它将焦磷酸盐的磷酸酐键中的自由能相互转化为钠和/或质子的动力。它们与旋转atp酶完全无关。它们存在于植物、原生动物寄生虫和（古）细菌中，但不存在于多细胞动物中，它们似乎在低能应激条件下是必不可少的：例如，敲除突变使原生动物寄生虫不具有传染性。它们的耦合机制基本上是未知的。我们的愿景是利用结构、单分子和功能研究来确定mPPase的精确作用机制，作为开发靶向分子的必要第一步。这项工作将对动物健康、粮食安全和人类疾病产生重要的长期效益。mpp酶存在于原生动物寄生虫中，如锥虫（昏睡病）、刚地弓形虫（感染高达90%的猪），更不用说恶性疟原虫（疟疾）了。这些疾病对世界广大地区的粮食安全和人类健康产生巨大影响，除疟疾外，所有这些疾病都被列为“被忽视”。此外，mppase也出现在普通拟杆菌中，这是脑脓肿的最常见原因。普通杆菌很难治疗，而且是抗生素耐药性的储存库，因为拟杆菌属具有极强的耐药性。我们的计划是使用多学科实验方法(即膜蛋白x射线晶体学，单分子荧光显微镜，快速电滴定法和最先进的快速光化学氧化/质谱法（FPOP/MS）与定向分子动力学结合在一起，确定具有利用瞬态作为药物靶点潜力的全范围运动。我们将解决不同类别的mppase的结构，特别是来自原生动物寄生虫和拟杆菌的mppase，以了解泵送的差异，并作为未来小分子抑制剂设计的基础。我们将使用单分子光谱来识别导致栅极打开和离子泵送的螺旋运动。快速电滴定法将确定电荷在膜上的运动动力学和水解动力学，FPOP/MS将以微秒级的时间分辨率识别TmPPase暴露表面的变化。所有这些工作都将整合到一个分子动力学模型中，以解释酶的功能，包括对实验无法实现的动力学状态结构的计算预测。了解“门打开”状态的结构将使下一阶段成为可能：识别使泵始终打开的分子。这些分子将成为高度特异性的候选药物。它们只会影响几类病原体，但对它们来说是完全致命的。

Even though they are targets for 60% of current drugs, integral membrane proteins account for less than 2% of the structures in the protein databank. Furthermore, fast kinetic studies on them have been mostly restricted to those with chromophores, like cytochrome c oxidase. This work will build on our ground-breaking x-ray structure of Thermotoga maritima Na+-pumping pyrophosphatase (TmPPase), published in 2012. Integral membrane pyrophosphatases (mPPases) are evolutionarily conserved novel "primary" ion pumps, interconverting the free energy in the phosphoanhydride bond of pyrophosphate into a sodium and/or proton motive force. They are completely unrelated to the rotary ATPases. They occur in plants, protozoan parasites and in (archae)bacteria but not in multicellular animals, and they appear to be essential under conditions of low-energy stress: knockout mutations render protozoan parasites non-infectious, for instance. Their coupling mechanism is essentially unknown. Our vision is to use structural, single molecule and functional studies to identify the precise mechanism of action in mPPase as the necessary first step for developing hit molecules. This work will have important long-term benefits for animal health, food security, and human disease.mPPases occur in protozoan parasites like Trypanosoma spp (Nagana, sleeping sickness), Toxoplasma gondii (infecting up to 90% of pigs), not to mention Plasmodium falciparum (malaria). These diseases have a huge impact on both food security and human health across wide swathes of the world, and all of them, with the exception of malaria, are classified as "neglected". In addition, mPPases also occur in Bacteroides vulgatus, which is the most common cause of brain abscesses. B. vulgatus is both very hard to treat and is a reservoir for antibiotic resistance because Bacteroides spp are extremely drug-resistant. Our plan is to use a multidisciplinary experimental approach (i.e. membrane protein x-ray crystallography, single-molecule fluorescence microscopy, fast electrometry and state-of-the-art fast photochemical oxidation/mass spectrometry (FPOP/MS) tied together with steered molecular dynamics to determine the full range of motions with the potential to exploit transient states as drug targets.We will solve structures of different classes of mPPases, especially ones from the protozoan parasites and Bacteroides, to understand differences in pumping and as the basis for future small molecule inhibitor design. We will use single molecule spectroscopy to identify motions in the helices leading to gate opening and thus ion pumping. The fast electrometry will determine the kinetics of charge movement across the membrane versus the kinetics of hydrolysis, and FPOP/MS will identify changes in the exposed surface of TmPPase with microsecond time resolution.All of this work will then be integrated within a molecular dynamics model to explain how the enzyme functions, including computational predictions of the structures of kinetic states that are inaccessible experimentally. Understanding the structure of the "gate open" state will enable the next stage: identifying molecules that keep the pumps always-open. Such molecules would be highly-specific drug candidates. They would affect only a few classes of pathogens, but would be completely lethal for them.