Structure Mechanics and Regulation Of The Vacuolar ATPase.

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.

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.