Dynamics and catalysis in 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.

Integral membrane pyrophosphatases (mPPases) are novel, conserved ion pumps that use the free energy in the pyrophosphate POP bond to generate a sodium and/or proton motive force. Although not found in multicellular animals, they occur in plants, protozoan parasites (eg malaria: P. falciparum) and (archae)bacteria and are essential under conditions of low-energy stress. We showed that mPPase is essential in P. falciparum (Totanes, unpubl.), and that the mechanism appears to be "binding change" (Li et al, Nat Comm, 2016). However, our most recent work shows that the enzyme is allosteric with half-site reactivity, and we have developed allosteric inhibitors (Vidilaseris et al, Sci Adv, 2019). Our model system is T. maritima mPPase (TmPPase).

Our main objectives are thus:
1. Solving structures of integral membrane proton-pumping pyrophosphatases with different inhibitors bound and from different families, including the Na/H-pumping organisms to identify (1) the structural correlates of K-dependence/independence and (2) the structural correlates of the pumped ion (Na/H/both).

2 We have Cys mutants that report on helix motions. We will use them with PELDOR and single molecule TIRF to observe changes +/- ligands, in lipids, nanodiscs to understand changes. smFRET will allow us to examine structural dynamics. Serial synchrotron crystallography will report on global changes upon ligand binding (TmPPase is slow at 20C). We will use steered molecular dynamics to derive models of transient states. The work will report on the allosteric changes that are part of our model of dual-pumping and will lead to new designs for inhibitors, covering all the expected timescales (ns-s).

This work will have important benefits for understanding allostery in mPPases and so in integral membrane proteins in general. By helping our design of mPPase inhibitors against protozoan parasites (in parallel work), the work will have benefits for animal health, food security and human disease.

The immediate beneficiaries of this research will be other academics, as outlined under academic beneficiaries. Non-academic beneficiaries fall into the following five main classes:
1) The public, through training of the next generation of scientists.
2) Society and the economy, through improved global health and food security
3) The private sector, through novel product development and commercial revenue
4) Public stakeholders
5) Society, through public engagement and discussion of science.

We focus on understanding the dynamics and allosteric mechanism of a novel membrane protein, integral membrane pyrophosphatase (mPPase). By doing so, we will (1) understand how our allosteric inhibitors work and (2) in the long-term improve them into potential lead molecules with low nM affinities. mPPases, which do not occur in mammals nor in most bacteria, are essential under conditions of low-energy stress in e.g. protozoan parasites. Protozoan parasites are major causes of both animal and human morbidity and mortality, through diseases like malaria (Plasmodium spp: 214M cases in 2015) and Toxoplasma gondii (infection rates as high as 90% in pigs). Many are on the WHO list of neglected tropical diseases.

1. Developing highly skilled people. A major transferrable benefit will be the people trained during the project. A PDRA trained on my previous BBSRC project is now employed at Peak Proteins. These include the PDRA, who will acquire multiple specialist scientific skills to use in research-based biotechnological industry and academia, the graduate and undergraduate students who will be involved in the projects, and the technician. The University of Leeds has staff development programmes to provide transferrable skills. These trained people, as they move to other institutions in academia, in government and in industry, will affect larger society positively.

2. Global health and food security. Novel drugs to treat protistal diseases could significantly improve global human and animal health by providing more treatment options. Reducing the human disease burden allows individuals to remain economically active and reducing the animal disease burden improves food security though reduced losses of animals.

3. Industrial involvement. SMEs and big pharma will benefit from this research. AG collaborates with Novartis and is part of two separate EU Innovative Training Networks including Novartis, AstraZeneca, Biomerieux and more than 10 SMEs. Infectious and parasitic diseases are a growing burden, so fundamental research on developing new targets and potential inhibitors will be exciting for companies for use both in animal and human health. This is relevant not only for protozoan diseases but also in treating Bacteroides brain abscesses, with an associated mortality of 20%. The timeframe for development is about 10 years.

4. Public health stakeholders. Leeds has an exemplary record in disseminating research and contributing to the public understanding of science in England and in Europe. New approaches to these diseases is important for national and international stakeholders, ranging from the Department of Overseas Development to international health charities and WHO.

5. Society. Work with potential to lead to superior outcomes will be disseminated widely (TV, radio, YouTube, press releases, Blogs, Twitter). MD simulations are particularly useful for science communication and will be used to explain findings to a general audience. Our focus is also on enthusing and training the next generation of scientists being STEM (Science Technology Engineering and Maths) ambassadors. We engage with students in secondary education; we aim to enthuse school children to study science in annual Discovery Zone workshops; and we inspire undergraduates completing 3rd-year projects.