The
devil so often is in the details. For proteins that orchestrate the
molecular business of life, there are devils and angels in their
details, down to the proteins’ constituent atoms. It’s at that level of
structural minutiae where the balance of health and disease, even life
and death, can pivot.
Published
in the journal Cell, a collaboration of nephrologists and
neuroscientists showed the value that emerges from uncommon alliances.
They and colleagues elsewhere reveal for the first time a portrait of
a life-and-death protein with enough clarity to finally reveal how it
works: as a minuscule ferry for molecular passengers that must cross
nearly a trillion cell membranes in tissues and organs ranging from
kidneys and brains to the inner ear and the lungs’ alveoli.
“With
new mechanistic understanding of this key protein, and how mutations in
it can shut it down, we are hoping that follow-on research will uncover
novel targets for treating kidney and brain diseases,” said a
corresponding author on the paper.
The
authors envision that the new high-resolution protein structures will
point toward therapeutic leads for treating diseases as prevalent as
acute kidney injury (affecting more than 4 million patients per year in the USA), chronic kidney disease (affecting some 800 million people worldwide) and Alzheimer’s disease (affecting an estimated 32 million people globally), and as rare as Donnai-Barrow syndrome (affecting fewer than 1,000 Americans), a genetic disorder with multiple physical and cognitive consequences.
The
protein is known as LRP2, a member of a family of LRP proteins found in
creatures ranging from worms to people. Compared to most proteins on
cell membranes, LRPs are huge, so much so that the scientists who
discovered LRP2 in the early 1980s, Marilyn Farquhar and Dontscho
Kerjaschki, dubbed it megalin. Some LRPs are built from more than 4,600
amino acids, the molecular building blocks of all proteins.
In
kidney cells, LRP2 is crucial for recovering reusable molecules from
filtered metabolic wastes from bodily fluid and so the body does not
have to spend energy and resources to make them again. For each of these
cells, there are likely tens of thousands of LRP2 proteins, distributed
on the surface like seeds on a strawberry.
“The
kidney is faced with recovering 99 percent of the body’s salt and water
that passes through the organ’s filters, and also with recovering 100
percent of small proteins that otherwise would dump into the urine and
out of the body,” said the author. “There have been general ideas about
how this recovery works, but its specificity has now been solved.”
Membrane
proteins like those in the LRP family are notoriously difficult to
isolate, let alone map out in detail. The lead author navigated a giant
step around that impasse with an arduous process in which he deployed a
bevy of biochemical techniques.
With
a mix of benchtop skill, creativity and determination, the lead author
harvested enough LRP2 protein from 500 mouse kidneys to solidify the
protein into a sample of sufficient size and purity for analysis with
advanced microscopy techniques. In the harvesting of the LRP2 molecules,
the author pulled off a biochemical tour de force: capturing LRP2
proteins locked into two of their key conformations, a crucial
laboratory feat for unveiling the protein’s machine-like actions in
cells.
With
a two-story, liquid-nitrogen-cooled, cryogenic electron microscope, the
authors collected vast amounts of structural data using hard-won LRP2
samples. Then, with the deft use of powerful computational tools to make
sense of the data, the researchers produced 3D protein structures in
near-atomic detail.
“We
now have the best 3D maps of the LRP2 protein ever created,” said a
co-author. With those maps, the authors could begin to tease out the
remarkable mechanism by which LRP2 works in cells.
One
of the mapped-out conformations captures the shape of the LRP2 when it
resides on and within a cell membrane. That’s where the protein picks up
molecular passengers from the liquid outside the cell–whether from the
urine produced in the kidney or the liquid around brain cells. Among
those passengers are small proteins, including tau and amyloid-beta
(both implicated in Alzheimer’s disease), insulin and ones that shuttle
vitamins A and D around cells.
The
other LRP2 conformation is the one the protein snaps into after it
becomes enveloped in a bit of cell membrane and ferries off to locations
inside the cell. It is in these little capsules, endosomes, where the
shape-shifting protein either enables its molecular passengers to be
recycled intact for further use or to be deconstructed into reusable or
disposable components.
From
this pair of LRP2 structures, the team was able to determine that the
protein undergoes a machine-like toggle between its passenger-embarking
form and its passenger-disembarking form. When LRP2 proteins are
mutation-free, they succeed in maintaining molecular balances in, for
example, blood and brain tissue. But when there are even tiny tweaks in
LRPs’ enormous molecular anatomy, these proteins can contribute to
disease.
In
kidney cells, for one, faulty LRP2 proteins renege on their normal task
of retrieving proteins that would otherwise be lost in the urine. That
can lead to a variety of conditions including chronic kidney disease,
Donnai-Barrow syndrome and conditions that are lethal in neonatal
stages.
In
the brain, LRP2 (and the related LRP1) normally help clear a variety of
toxins, among them tau protein fragments, which have long been
associated with Alzheimer’s disease. But proteins of the LRP family also
have been shown to transfer such fragments between brain cells,
potentially contributing to the disease process.
“You could imagine that trying to inhibit this from happening with a drug could be helpful,” said a corresponding author.
This is where the power of cryogenic-EM comes in strong.
“If
you know exactly the atomic details of where the tau binds, you might
actually block that using either an antibody or a small molecule,” said a
co-author. “Cryo-EM can get you to the level of detail you need in
order to work on potential new therapies.”
“This
is the beginning of a long road of discovery of how these LRP proteins
work and to new drug targets for a range of diseases,” said a
corresponding author.
https://www.cell.com/cell/fulltext/S0092-8674(23)00046-6