Exploiting the structure of integral membrane pyrophosphatases

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.

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 occur in plants, apicomplexan 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 apicomplexan parasites such as Toxoplasma and Trypanosoma 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 specific inhibitors. This work will have important benefits for animal health, food security, and human disease since e.g. protozoan parasites like Trypanosoma spp (Nagana, sleeping sickness), Toxoplasma gondii (infecting up to 90% of pigs), not to mention Plasmodium falciparum (malaria) cause serious diseases

Our overarching vision is to understand the complete atomic mechanism of integral membrane ion-pumping pyrophosphatases as the necessary first step for developing drug-like molecules. Our main objectives are thus:
1. Solving structures of integral membrane proton-pumping pyrophosphatases from disease causing organisms (Bacteroides and apicomplexa)

2. Complementing the structural information through fast kinetics and single molecule work to gain insight into the transient "gate-open" species that leads to pumping. Molecular dynamics simulations that give testable information about "gate-open" and other transient states and provide hypotheses about the basis of ion-pumping specificity (proton versus sodium versus both).

The main techniques we will use are x-ray crystallography, single molecule fluorescence microscopy, electrometry, fast photochemical oxidation/mass spectrometry and steered molecular dynamics.

The immediate, short-term beneficiaries of this fundamental research will be other academics, as outlined in the academic beneficiaries section. However, in the medium to long term, the non-academic beneficiaries of this work fall into three main classes:
1) The wider public, through improved global health and food security
2) The commercial private sector, through improved options for novel product development and commercial revenue
3) Charities within the non-public sector

This proposal focuses on structural and functional understanding of a novel membrane protein, the integral membrane pyrophosphatase (mPPase). mPPases do not occur in mammals nor in most bacteria, but are essential under conditions of low-energy stress in both protozoan parasites and the anaerobic opportunistic pathogen Bacteroides. This project will lay the groundwork for future development of molecules that target these pathogens highly specifically. Protozoan parasites, such as P. falciparum (malaria: 207M cases in 2012) and Toxoplasma gondii (infection rates as high as 90% in pigs), are major contributors to both animal and human morbidity and mortality. Many are on the WHO list of neglected tropical diseases.

Global health and food security. Being able to target specific proteins that are unique to the pathogenic organisms will reduce the problem of drug resistance: only the "bad" species is targeted, so there is no selection pressure on other organisms to acquire and spread resistance.

Industrial involvement. SMEs and big pharmaceutical companies will benefit from this research. Researchers at Leeds work with companies, including MedImmune and GlaxoSmithKline; and AG has had grant funding from Merck and collaborates with Novartis and the California SME ActivX. It is widely accepted that infectious diseases are no longer a thing of the past. This work will present new targets for the companies for use both in animal and human health, and will be relevant not only to protozoan diseases but also in treating Bacteroides brain abscesses, with an associated mortality of 20%. The timeframe for development is 5-15 years but this work provides the fundamental research on which it will be based.

Academic stakeholders. We will communicate with academic stakeholders by giving high-visibility seminars at major conferences and universities, and by publishing our research in the very best journals.

Developing highly skilled people. A major transferrable benefit of all academic research is the people trained during the project. The PDRA on the project will acquire multiple specialist scientific skills (membrane protein crystallography, TIRF, FPOP/MS) to use in research-based biotechnological industry and academia. In addition the University of Leeds has an extensive career development program that will provide transferrable skills. These trained people, as they move to other institutions in academia, in government and in industry, will affect the larger society positively.

Public health stakeholders. We have a strong track record in disseminating their 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.

Reaching the general public. Work with potential to lead to superior outcomes will be disseminated widely (TV, radio, YouTube, press releases). Atomistic 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. We will also continue to engage with students in secondary education, by inspiring visiting UCAS students, by annual Discovery Zone workshops to enthuse school children to study science and by being STEM (Science Technology Engineering and Maths) ambassadors.