Science Show & Tell: New insight into how cells control the pH of their internal compartments

Dr. John Rubinstein, Senior Scientist in Molecular Structure & Function, has a study called 'Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase' published in the October 24, 2016 Advance Online Publication of Nature. We sat down with him to learn more about his research:

What did your research find?

We have determined the first atomic structure of part of a V-ATPase called the VO region. V-ATPases are enzymes that are embedded in membranes in cells: they use the chemical energy currency of cells, called ATP, to pump protons across the membrane and control the pH of internal compartments of all cells in higher organisms (e.g. animals, plants, and fungi). Some specialized cells in our bodies also use V-ATPases to control the pH of their external environment. Maintaining different parts of the cell at their correct pH at the correct time is essential for many processes that have to happen in a healthy cell. Examining this structure now shows us how V-ATPases use energy from ATP to pump protons.

How is this research significant?

We have determined the first atomic structure of part of a V-ATPase called the VO region. V-ATPases are enzymes that are embedded in membranes in cells: they use the chemical energy currency of cells, called ATP, to pump protons across the membrane and control the pH of internal compartments of all cells in higher organisms (e.g. animals, plants, and fungi). Some specialized cells in our bodies also use V-ATPases to control the pH of their external environment. Maintaining different parts of the cell at their correct pH at the correct time is essential for many processes that have to happen in a healthy cell. Examining this structure now shows us how V-ATPases use energy from ATP to pump protons.

This research answers a question that people have been trying to answer for more than 25 years. V-ATPases, along with related enzymes called ATP synthases, are remarkable because they have spinning parts that link the process of using ATP to the movement of protons across a membrane. One of my PhD supervisors shared the Nobel prize in Chemistry in 1997, largely for proving that this rotation occurs in ATP synthases and showing how rotation drives ATP production in the part of the enzyme that sticks out of the membrane. His group did that by determining the structure of that part of the enzyme using X-ray crystallography. However, how rotation is linked to the movement of protons across the membrane has remained a mystery because, until now, no one had been able to determine the structure of the membrane-embedded region of one of these enzymes at high resolution.

How can this basic science finding be applied?

There isn’t just one form of V-ATPase, but actually many subtly different forms in different locations in cells. For example, osteoclasts, which are cells that are constantly taking up bone minerals during bone maintenance, use a specific form that is different from the form used in the part of the cell where new proteins are produced. Pathogenic fungi, which cause serious infections in immunocompromised people, rely heavily on their V-ATPases, which differ subtly from the human forms. Now that we have the basic structure for a V-ATPase and have learned how to determine these structures, we want to compare the atomic structures of the different V-ATPases. That comparison will help us design therapeutics that could selectively turn off V-ATPases, such as turning down the osteoclast V-ATPase when bone minerals are being removed too quickly. Specifically blocking the fungal V-ATPase would help treat fungal infections.

Tell us more about the technology you use to see enzymes at an atomic level.

We used a technique called single particle electron cryomicroscopy or cryo-EM. In cryo-EM you just isolate the molecules you’re interested in and image them with an electron microscope frozen in a thin film of ice. You then use computer algorithms to determine the 3-D structure of the molecules from those images. Along with others, we’ve been using cryo-EM and developing new cryo-EM methods for quite a few years. However, the technique recently went through a “resolution revolution” when new highly-sensitive cameras for electron microscopes were introduced, suddenly allowing cryo-EM to determine atomic-resolution structures that would have been impossible to determine a few years ago. It’s an extremely exciting time in molecular biology because of this technology, and we’re preparing to install a new high-resolution high-throughput cryo-EM microscope at SickKids.