First detailed look at how molecular Ferris wheel delivers protons to
cellular factories

Date:
October 7, 2020
Source:
DOE/SLAC National Accelerator Laboratory
Summary:
All cells with nuclei, from yeast to humans, use molecular machines
called proton pumps to regulate the acidity of organelles -
compartments where various types of work are done. A new study
reveals a key step in how these Ferris wheel-like pumps operate.



FULL STORY ==========================================================================
All cells with nuclei, from yeast to humans, are organized like cities,
with a variety of small compartments -- organelles -- that serve as
factories where various types of work are done. Some of those factories,
like the ones that break down and recycle molecules, need to continually
pump in protons - - hydrogen atoms with their electrons stripped off --
to maintain the acidic environment they need to do their job. For this
they rely on molecular Ferris wheels.


========================================================================== Embedded in the organelle's fatty outer membrane, these microscopic
machines have rotors that spin 100 times per second, picking up protons
from outside the organelle and dropping them off on the inside.

Now scientists have figured out a key step in how these Ferris wheels
work in a yeast proton pump known as vacuolar ATPase (V-ATPase). The
results of their study, which combined high-resolution images made at
the Department of Energy's SLAC National Accelerator Laboratory with supercomputer simulations, were published in Science Advances today,
giving scientists insight into a fundamental process that could
potentially be harnessed to thwart disease.

"The V-ATPase proton pumps perform a wide range of functions, from helping transmit nerve signals to helping specialized cells secrete acid for maintaining bone," said Stephan Wilkens, a biochemist at SUNY Upstate
Medical University and study co-author. "Malfunctions in these molecular machines contribute to diseases such as osteoporosis, neurodegeneration, diabetes, cancer and AIDS, so understanding them is important for human health." Wah Chiu, a professor at SLAC and Stanford and co-director of
the Stanford-SLAC Cryo-EM Facilities where the imaging was done, said scientists are already investigating how these pumps in human cells might affect replication of the COVID-19-causing virus in patients. "It turns
out the majority of therapeutic drugs on the market target molecular
machines like this one that sit in cell membranes," he added.

Watching the wheel go round No human cell can function without proton
pumps, which among other things help organelles intercept viruses and
other pathogens and divert them to cellular trash bins.

While previous studies had determined the molecular structure and
basic function of V-ATPases in a number of organisms, Wilkens said,
"the big question was how do they work? To explain the mechanism it's
helpful to see it in action, just like the first serial snapshots of a galloping horse finally settled the question of whether it always had
at least one hoof on the ground.

The answer was no." In earlier cryo-EM research, Chiu, Wilkens, SLAC/
Stanford postdoctoral researcher Soung-Hun Roh and others produced high-resolution images that allowed them to identify the 10 amino acid
"seats" on the yeast Ferris wheel that bind protons and carry them
through the membrane to the organelle's interior, as well as other
amino acids that catch them when they arrive. Based on that picture,
they suggested that the proton drop-off might be aided by water molecules,
but their images were not sharp enough to confirm that the water molecules
were there.

In the current study, thanks to another round of even higher resolution
cryo-EM imaging at SLAC they were able to locate the water molecules
around the suspected proton path. To make the proton pump motor come
to life, a research group led by Abhishek Singharoy at the Arizona
State University Biodesign Institute developed computer simulations of
the process and ran them on a DOE supercomputer at Oak Ridge National Laboratory.

The simulations, which incorporated cryo-EM structures derived from
images of the yeast Ferris wheel captured at two different points in
its rotation, confirmed the experimentally observed water molecules
lining up to form "wires" at the proton drop-off point. These wires
convey protons from their seats on the Ferris wheel to landing spots
inside the organelle, like a fire brigade passing buckets hand to hand, bridging a gap they couldn't navigate on their own.

Going forward, Chiu said, recent advances in cryo-EM that allow imaging of individual particles at atomic resolution -- even when they take slightly different shapes -- will open new opportunities for using it as a tool
to discover effective drugs for illnesses involving proton pumps.


========================================================================== Story Source: Materials provided by
DOE/SLAC_National_Accelerator_Laboratory. Original written by Glennda
Chui. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Soung-Hun Roh, Mrinal Shekhar, Grigore Pintilie, Christophe Chipot,
Stephan Wilkens, Abhishek Singharoy, Wah Chiu. Cryo-EM and MD infer
water-mediated proton transport and autoinhibition mechanisms of
Vo complex. Science Advances, 2020; 6 (41): eabb9605 DOI: 10.1126/
sciadv.abb9605 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/10/201007145304.htm

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