Dynamics and catalysis in integral membrane pyrophosphatases

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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.