Structure Mechanics and Regulation Of The Vacuolar ATPase.

Lead Research Organisation: University of Leeds
Department Name: Institute of Membrane & Systems Biology

Abstract

The vacuolar ATPase (V-ATPase) is a complex multi-component protein found in virtually all living cells. It is a miniature mechanical rotary motor, only thirty millionths of a millimetre long but capable of pushing protons into or out of specialised compartments in the cell, regulating acidity. The V-ATPase is a 3-stroke motor that uses ATP as a fuel to rotate a central axle, which is linked to a 10-stroke acid pump plugged into the cell membrane. A molecular gearbox links the two motors allowing it to push protons against different gradients. However, the way in which the motors work and are controlled by the cell are still unclear. This project aims to generate a high resolution structure, through electron microscopy and X-ray crystallography, revealing the composition and mechanism of this motor and its gearbox. Understanding the structure and mechanism of this motor will provide important information which can be used to develop new drugs, important because of the role the protein plays in a number of diseases including osteoporosis, kidney disease and cancer. By understanding the structure, mechanism and regulation of the protein we may be able to develop novel ways of switching it off with drugs in diseased tissue.

Technical Summary

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump essential for the function of virtually all eukaryotic cells. It is a membrane-bound rotary motor, larger and more complex than the related ATPase synthase, and is responsible for acidifying intracellular compartments, energising membrane transport and pH homeostasis. Mutations in V-ATPase genes cause osteopetrosis, kidney disease and autophagic myopathy, and its involvement in osteoporosis and metastatic cancer means that controlling its activity with drugs has therapeutic potential. Despite its importance, our understanding of the V-ATPase mechanism and regulation is poor and is a major block in therapeutic development, since structural data often underpins therapeutic drug design. This project aims to produce a high resolution structure of the native V-ATPase (at resolution <10?), combining techniques in single particle cryo-electron microscopy and X-ray crystallography. This will allow for previously unresolved features of this rotary motor family to be seen. In particular it will provide information about the organisation of subunit a, which is poorly defined in both the F and V-ATPase family and is a target for a number of potent inhibitors. This will be complemented by both kinetic analysis and time-resolved cryo-EM, to trap the V-ATPase in conformational states, providing insights into the rotary mechanism. This complex motor is regulated in response to physiological signals indicating low energy, leading to reversible domain dissociation. The nature of the structural changes that occur in response to these signals will also be studied by single particle cryo-EM and, where appropriate, crystallography. Time-resolved single particle cryo-EM is the only general purpose method that can give high spatial (<10?) and time (<5ms) resolution, but despite the power of this technique it is infrequently used. I aim to further develop the general utility of this technique and become a leading expert in what promises to become an important general-purpose method in structural biology. A proven key to success in structural biology is having access to the right material. By having access to an excellent V-ATPase preparation which is homogeneous, stable and able to be highly concentrated, combined with access to one of only a handful of time-resolved apparatus worldwide, I am uniquely placed to carry out this work. This proposed research will further our understanding of a complicated molecular motor and will aid the development of the time-resolved cryo-EM technique. Greater understanding of the structure, mechanism and regulation of the V-ATPase may provide a springboard for therapeutic design.

Publications


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Bockelmann S (2010) Archazolid A binds to the equatorial region of the c-ring of the vacuolar H+-ATPase. in The Journal of biological chemistry
Muench SP (2011) Structural divergence of the rotary ATPases. in Quarterly reviews of biophysics
Rawson S (2015) Structure of the vacuolar H+-ATPase rotary motor reveals new mechanistic insights. in Structure (London, England : 1993)
Rawson S (2016) Rotating with the brakes on and other unresolved features of the vacuolar ATPase. in Biochemical Society transactions
 
Description Development of time-resolved methodologies with Prof Howard White 
Organisation Eastern Virginia Medical School (EVMS)
Country United States of America 
Sector Academic/University 
PI Contribution Prof Howard Whte has been visiting Leeds for the past 4 yeasr to help develop the time-resolved EM setup. The last two years has seen significant development of the system. I have been responsible for testing the EM grids and optimising the setup.
Collaborator Contribution Prof Howard White has brought technical support in the form of engineering expertise to develop the required hardware.
Impact This has now resulted in the submision of a BBSRC methods development grant. It has also sparked a new collaboration with Dr Martin Trebbin in Hamburg who currently develops the XFEL beamline time resolved system.
Start Year 2012
 
Description Dr Sjors Scheres Data Processing 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Provide data which is problematic with conventional electron microscopy data processing programs.
Collaborator Contribution Dr Sjors Scheres has provided valuable help in using the Maximum Likelihood data processing suite
Impact One paper is currently under review and further publications are planned
Start Year 2010
 
Description Working with a group in Osnabrueck, Germany on V-ATPase mechanism and regulation 
Organisation University of Osnabrück
Department School of Biology/Chemistry Osnabrück
Country Germany, Federal Republic of 
Sector Academic/University 
PI Contribution The group in Osnabrueck have a depth of experience and my team work closely with them to look at the structure through EM and X-ray crystallography. A paper was published last year where I was involved in developing models of the c-ring to allow us to study the binding of the Archazolid inhibitor (PMID: 20884613). We are currently collaborating on many different aspects of V-ATPase functionality, including subunit glycosylation, domain structures and looking at a new inhibitor family.
Collaborator Contribution Supplying me with V-ATPase sample which is extracted from the Manduca caterpillar. Provide biochemical data on inhibitor binding and characterisation studies
Impact PMID: 19244615 (cryo-electron microscopy of the V-ATPase motor reveals its mechanical and regulatory complexity)(Carried out before MRC funding awarded) PMID: 20884613 (Archazolid A binds to the equatorial region of the c-ring of the V-ATPase). This is a multi-disciplinary team with my role being primarily to carry out structural studies (X-ray crystallography, EM and structure modelling) with biochemical characterisation being carried out in Osnabrueck
Start Year 2007
 
Description A-level student Lab visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 4 A level students were invited to spend a week in the laboratory (2 per week). They gained experience at data processing and electron microscopy but more importantly gained skills in data and time management and enjoyed experiencing the University atmosphere. This helped focus their degree choices.

This was a provisional test to forge links between Beckfoot secondary school and the University of Leeds. It was a great success and we can now offer the 2 best A-level students in Science the opportunity to visit the lab for 1 week. I am also helping the school and others soon with giving career advice
Year(s) Of Engagement Activity 2012
 
Description Discovery Zone 
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 Over 200 pupils visited the Discovery zone at which I was responsible for running two stalls. One on protein structure one on ion channels. These were both interactive and the pupils were engaged in the activities. The success of this means that it will be run again next year.

We received excellent feedback from teachers and pupils who found the event to be both informative and fun. It is hoped that this will encourage more students into a science profession
Year(s) Of Engagement Activity 2013,2014,2015,2016
URL http://www.fbs.leeds.ac.uk/outreach/schools/lfos.php
 
Description Laboratory visit by A-level pupils 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact As part of a continuing scheme with Beckfoot School, I allowed one pupil (decided by an application procedure) to visit the laboratory for a week. He took part in a range of experiments and gained a lot of insights into both a scientific career and the University environment

It firmed up the students belief that he would like a career in science and allowed him to understand better the different areas of research available.
Year(s) Of Engagement Activity 2013,2014
 
Description Public engagement event at the Astbury Conversation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Organised the public engagement part of the Astbury conversation with ~20 stalls. Also designed and ran my own stall in addition to co-ordinating the event. The stalls were of a wide remit with information on how proteins work, electron microscopy, protein weaving etc....
Year(s) Of Engagement Activity 2016
URL http://www.astburyconversation.leeds.ac.uk/
 
Description School Visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A presentation was given to the A-level students at Beckfoot academy on careers in and out of science but involving a science degree. This was followed by 1 on 1 discussions with several pupils on career choices and university applications.

This has now turned into a 6 month event where I go in to talk to the students and give advice. I have also arranged for 4 students to visit the laboratory over the summer. These are to be selected on a competition basis so the best students based on ability can visit.
Year(s) Of Engagement Activity 2011
 
Description summer placement 
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 2 summer students gained 8 weeks experience in the laboratory during the summer of 2014. Both will use their experience in applications for PhD places this year

Resulted in scientific output, generation of pilot data and gave two undergraduates invaluable research experience
Year(s) Of Engagement Activity 2014