Exploiting the structure of integral membrane pyrophosphatases

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We work on integral membrane pyrophosphatases, which occur in bacteria, archaebacteria, plants, and protozoan parasites that cause disease like malaria and leishmania - but that do not occur in humans or other animals. They are thus potential drug targets. The enzymes create charge gradients that enable the cells to function. We have discovered how the enzyme works at the molecular level, and we published that in Nature Communications in 2016. This work describes two different enzymes, one from plants and one from an archaebacterium. In addition, we have begun drug design and screening studies, and have been able to find about 5 compounds that appear to inhibit the enzyme at micromolar level in tests. One of these compounds also kills the malaria parasites in the red blood cell assay, so it is actually a potential new drug that we now need to optimise. It has the great advantage over many other starting compounds in that there is no reason to believe that it will bind to human proteins, because there are no similar proteins to the integral membrane pyrophosphatase in the human body. We have now solved the structures of an inhibited form of the Thermotoga enzyme (manuscript published) as well as structures of three different enzymes, from P. aerophilum, Bacteroides vulgatus and Clostridium leptum. All of this work will also be published. We have also developed a series of novel inhibitors that we are optimising (one manuscript published).
Exploitation Route Other groups can use the work to develop pyrophosphate-based batteries, which could be solar-powered. In addition, other groups both here and in Finland are exploiting the structures to develop novel compounds that may become a novel class of antimalarial drugs.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The findings have been used to develop a new method of stabilising membrane proteins that is currently licensed by Peak Proteins. Further licensing is being explored
First Year Of Impact 2019
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description Deans Vacation Scholarship Award for Steven Harborne
Amount £2,500 (GBP)
Organisation University of Leeds 
Sector Academic/University
Country United Kingdom
Start 06/2017 
End 08/2017
 
Description Dynamics and catalysis in integral membrane pyrophosphatases
Amount £598,307 (GBP)
Funding ID BB/T006048/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2020 
End 01/2023
 
Description Global Challenges Research Fund
Amount £83,000 (GBP)
Funding ID CH160038 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 02/2018
 
Description Marie Curie Innovative Training Network
Amount € 3,243,300 (EUR)
Organisation European Union 
Sector Public
Country European Union (EU)
Start 04/2017 
End 03/2021
 
Description Marie Curie postdoctoral fellowship
Amount € 183,000 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 09/2016 
End 08/2018
 
Description Marie Curie postdoctoral fellowship
Amount € 183,000 (EUR)
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 07/2015 
End 06/2017
 
Description Maximising Academic Translational Projects Towards Optimal Commercialisation Outputs and Impact
Amount £359,996 (GBP)
Funding ID MC_PC_18053 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2019 
End 03/2021
 
Description Open application
Amount € 863,000 (EUR)
Organisation Jane & Aatos Erkko Foundation 
Sector Charity/Non Profit
Country Finland
Start 09/2015 
End 08/2018
 
Description Proximity to discovery industrial placement award for Steven Harborne
Amount £3,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 12/2017
 
Description Proximity to discovery pump-priming award for Steven Harborne
Amount £5,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2017 
End 12/2017
 
Description White Rose Studentship
Amount £14,777 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description White Rose graduate fellowship scheme
Amount £45,000 (GBP)
Organisation White Rose University Consortium 
Sector Academic/University
Country United Kingdom
Start 08/2015 
End 07/2018
 
Title New Approach for Engineering proteins 
Description We have developed a novel way of designing and testing protein mutations, using a statistical approach called "Incomplete Factorial". This enables one to identify useful mutations to a protein in a complex way more efficiently than by scanning mutagenesis, and provides a way of identifying and using 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact We have been able to use it to develop a variant of the E. coli protein AcrB that does not bind to Nickel-resin. This enables more membrane proteins to be efficiently purified in E. coli, thus reducing the need for expensive insect cell/mammalian cell approaches. 
 
Title Rapid method for determining pyrophosphatase activity 
Description We have developed a PAGE-based version of the classical colourimetric assay for pyrphosphatases (PPases). The assay in solution uses a molybdate-based reaction to form a colour compound that can be detected at 700 nm. We were able to determine that it is possible to run the same assay in PAGE and detect using conventional gel imaging, providing a way of determining amounts of active protein before final purification. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact We have been using it to optimise membrane protein purification of various integral membrane pyrophosphatases. It has been used in new papers from the laboratory, and one is in preparation describing the method. 
 
Title New approach for rational mutagenesis of membrane proteins 
Description We have developed a novel way of designing and testing protein mutations, using a statistical approach called "Incomplete Factorial". This enables one to identify useful mutations to a protein in a complex way more efficiently than by scanning mutagenesis, and provides a way of identifying and using mutations that individually are deleterious , but together are beneficial. 
Type Of Material Data analysis technique 
Year Produced 2018 
Provided To Others? Yes  
Impact We have used this to modify E. coli AcrB so that it does not bind nickel-resin any more. The precise combination of mutations required could not have been foreseen. We have used it to generate an E. coli strain to allow more efficient membrane protein purification. 
 
Title PDB codes: AG lab 2015-on 
Description The following is a list of the PDB codes from the lab since 2015. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Structures used for drug design, modelling etc. 6QXA, 6QP4, 6AFS, 6AFT, 6AFU, 6AFV, 6AFW, 6AFY, 6AFZ, 5NBQ, 5MN2, 5ML9, 5LZQ, 5LNL, 5LZR, 5GPJ 
 
Description Collaboration on integral membrane Pyrophosphates 
Organisation University of Helsinki
Department Division of Pharmaceutical Chemistry and Technology
Country Finland 
Sector Academic/University 
PI Contribution Provide protein, intellectual input into possible modifications, binding studies
Collaborator Contribution Provide novel inhibitor design, intellectual input into new drugs, testing of new molecules in Plasmodium falciparum and animal models
Impact Goldman, A., af Gennäs, G. B., Xhaard, H., Meri, S. & Yli-Kauhaluoma, J. Pyrofosfaatti lääketieteessä. Pyrofosfaatti lääketieteessä. Duodecim 132, 1111-1117 (2016). Shah, N. R., Vidilaseris, K., Xhaard, H. & Goldman, A. Integral membrane pyrophosphatases: a novel drug target for human pathogens? AIMS Biophys 3, 171-194 (2016). Vidilaseris, K., Kellosalo, J. & Goldman, A. A high-throughput method for orthophosphate determination of a thermostable membrane-bound pyrophosphatase activity. Anal Meth 10 646-651 (2018). Harborne, S. P. D., Strauss, J., Turku, A., Watson, M. A., Tuma, R., Harris, S. A. & Goldman, A. Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET. Methods Enzymol. 607, 93-130 (2018). Strauss, J., Wilkinson, C., Vidilaseris, K., Harborne, S. P. D. & Goldman, A. A Simple Strategy to Determine the Dependence of Membrane-Bound Pyrophosphatases on K+ as a Cofactor. Methods Enzymol. 607, 131-156 (2018). Multidisciplinary collaboration Crystallography, protein chemistry, biophysics, enzymology, drug design, protein modelling
Start Year 2015
 
Description Collaboration on integral membrane Pyrophosphates 
Organisation University of Leeds
Department School of Chemistry Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Provide protein, intellectual input into possible modifications, binding studies
Collaborator Contribution Provide novel inhibitor design, intellectual input into new drugs, testing of new molecules in Plasmodium falciparum and animal models
Impact Goldman, A., af Gennäs, G. B., Xhaard, H., Meri, S. & Yli-Kauhaluoma, J. Pyrofosfaatti lääketieteessä. Pyrofosfaatti lääketieteessä. Duodecim 132, 1111-1117 (2016). Shah, N. R., Vidilaseris, K., Xhaard, H. & Goldman, A. Integral membrane pyrophosphatases: a novel drug target for human pathogens? AIMS Biophys 3, 171-194 (2016). Vidilaseris, K., Kellosalo, J. & Goldman, A. A high-throughput method for orthophosphate determination of a thermostable membrane-bound pyrophosphatase activity. Anal Meth 10 646-651 (2018). Harborne, S. P. D., Strauss, J., Turku, A., Watson, M. A., Tuma, R., Harris, S. A. & Goldman, A. Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET. Methods Enzymol. 607, 93-130 (2018). Strauss, J., Wilkinson, C., Vidilaseris, K., Harborne, S. P. D. & Goldman, A. A Simple Strategy to Determine the Dependence of Membrane-Bound Pyrophosphatases on K+ as a Cofactor. Methods Enzymol. 607, 131-156 (2018). Multidisciplinary collaboration Crystallography, protein chemistry, biophysics, enzymology, drug design, protein modelling
Start Year 2015
 
Description Collaboration on the Comatose ABC family D transporter 
Organisation Rothamsted Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Setting up for crystallisation of the comatose protein
Collaborator Contribution Production of the comatose protein
Impact none so far
Start Year 2014
 
Description Collaboration on the Comatose ABC family D transporter 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Setting up for crystallisation of the comatose protein
Collaborator Contribution Production of the comatose protein
Impact none so far
Start Year 2014
 
Description Collaboration with Nankai 
Organisation Nankai University
Country China 
Sector Academic/University 
PI Contribution Will provide clones and house a graduate student for the purpose of collaborative research
Collaborator Contribution Provides graduate student for two years to perform research on membrane proteins
Impact 10.1371/journal.pone.0143010
Start Year 2014
 
Description Collaboration with Turku 
Organisation University of Turku
Country Finland 
Sector Academic/University 
PI Contribution We have solved the structures of a number of pyrophosphatases and shared prepublication results with them over the years. We have had joint grants and students.
Collaborator Contribution The group of Reijo Lahti has contributed protein, clones and reagents to the collaboration
Impact eg: Heikinheimo, P., Tuominen, V., Ahonen, A.-K., Teplyakov, A., Cooperman, B. S., Baykov, A. A., Lahti, R. & Goldman, A. Toward a Quantum-mechanical description of Metal Assisted Phosphoryl Transfer in Pyrophosphatase. Proc. Natl. Acad. Sci. U S A 98, 3121-3126 (2001). Oksanen, E., Ahonen, A.-K., Tuominen, H., Tuominen, V., Lahti, R., Goldman, A. & Heikinheimo, P. A complete structural description of the catalytic cycle of yeast pyrophosphatase. Biochemistry 46, 1228-1239 (2007). Fabrichniy, I. P., Lehtiö, L., Tammenkoski, M., Zyryanov, A. B., Oksanen, E. J., Baykov, A. A., Lahti, R. & Goldman, A. A Trimetal Site and Substrate Distortion mark the active site of Family II Inorganic Pyrophosphatase. J. Biol. Chem. 282, 1422-1431 (2007). Tuominen, H., Salminen, A., Oksanen, E., Jämsen, J., Heikkilä, O., Lehtiö, L., Magretova, N. N., Goldman, A., Baykov, A. A. & Lahti, R. Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridium perfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate. J. Mol. Biol. 398, 400-413 (2010).
 
Description Structural studies of mutants of Mhp1 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Advice on crystallising the Mhp1 mutants; access to the crystallisation robotics
Collaborator Contribution production, purification, structure solution.
Impact Initial draft of a manuscript on the Mhp1 mutants
Start Year 2014
 
Description DiscoveryZone 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 300 keystage 2 and 3 pupils attend and are engaged in hands-on scientific activities from over 30 different stations.
Year(s) Of Engagement Activity 2016,2017,2018,2019
URL http://www.fbs.leeds.ac.uk/outreach/schools/lfos.php
 
Description Participation in open day activities associated with the Astbury Conversation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 300 members of the general public, including school-children were part of the open sessions at the Astbury conversation. The keynote speaker in 2018 was the Nobelist Brian Kobilka, and in 2020 will be Nobelist Richard Henderson. There was increased interest in the idea of using structural understanding to develop much improved drugs - for instance in the opioid family of drugs. We present a stall of the activities in the laboratory, including hands-on demonstrations
Year(s) Of Engagement Activity 2018,2020
 
Description Specific outreach for middle-school girls 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact About 5 girls from disadvantaged backgrounds attended an open-day we organised at the university. The purpose was to introduce them to the idea of "This girl can", so they met a variety of women in my laboratory from non-traditional backgrounds (non-British; working-class; coming back into education after time out; postgraduates etc), and had the chance to do some "hands-on" playing in the laboratory. There was a clear increase in related subject areas, and the idea that they could become scientists.
Year(s) Of Engagement Activity 2019
 
Description Student visits for the Baldwin memorial symposium 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 127 keystone 4 pupils from local schools attended a public lecture by the Nobelist Professor Sir John Walker, FRS, and also were involved in a visit to the laboratories. They had an introduction to scientific techniques, sparking interest in biological research areas.
Year(s) Of Engagement Activity 2015
URL http://www.fbs.leeds.ac.uk/baldwin/