June 09, 2005
Researchers Get First Peek at Amyloid's Spine
Howard Hughes Medical Institute researchers have provided the first
detailed look at the core structure of the abnormal protein filaments
found in at least 20 devastating diseases, ranging from Alzheimer's to
Creutzfeldt-Jakob disease, the human version of “mad cow”
disease.
The images reveal that the filaments form a short zipper that is
closed and stuck. To get a more realistic picture of what the fibrils
look like, however, one should picture a towering stack of zippers,
each of which is tightly bonded to the one below.
“To do something about these diseases, you have to be able to see the parts at the atomic level.”
David Eisenberg
The first atomic details of the interconnected protein segments were
reported in the June 9, 2005, issue of Nature.
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A molecular illustration shows the structure of the amyloid spine.... more
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In each disease, a different protein transforms into the misfolded
threads known as amyloid fibrils. Scientists believe that the various
proteins share a common underlying feature that explains how they
assemble into the persistent fibrils that can accumulate in the brain
and other tissues.
"To do something about these diseases, you have be able to see the
parts at the atomic level," said senior author David Eisenberg, a
Howard Hughes Medical Institute (HHMI) investigator at the University
of California, Los Angeles. "Only then can you design an
intervention."
The common trait of these different proteins was discovered more
than thirty years ago. But even the most advanced technologies have
been unable to capture anything more than a fuzzy image.
"We call it the spine of the amyloid," said Eisenberg, who is also
director of the UCLA-Department of Energy Institute of Genomics and
Proteomics. "A little bit of each protein forms the spine, and the rest
of the protein is hanging out in globular domains that decorate the
spine and give the fibril its thickness and bumpiness. Once these
amyloid fibrils form in tissues or cells, they are very hard to get rid
of."
Now, he and his colleagues report the first detailed look at one
protein's version of the shared core feature. In this case, it was a
yeast prion, a misfolded protein that has the additional knack of being
able to infect other cells or organisms, said first author Rebecca
Nelson, a graduate student. Unlike in people, the yeast prion causes a
condition which may be beneficial. In people, scientists do not know
the role of the fibrils in the disease process in most associated
diseases, but the formation of fibrils is associated with diseases.
According to yeast prion expert Jonathan Weissman, an HHMI
investigator at the University of California, San Francisco,
determining this structure “is a monumental achievement that will
open up a new era in the structural analysis of amyloids.”
The path to the discovery began several years ago, when co-author
Melinda Balbirnie had narrowed down the stretch of prion necessary for
fibrils to only seven amino acids, which were located at one end of the
entire protein. Filling a test tube with just those snippets was enough
to form short thin threads with the same essential characteristics of
the common amyloid spine, a structure known as a cross-beta sheet.
Once it begins, the structure of a growing amyloid fiber is
irresistible to other identical proteins or, as in this study, the
crucial peptide subcomponents. The fibril spine elongates as pairs of
the short beta-sheet segments stack up like teeth in a zipper.
When Balbirnie added more peptides to the test tube, she found that
microcrystals formed and dropped to the bottom of the test tube. The
crystals were about 50,000 times smaller than those normally used to
determine atomic details of protein architecture. The researchers tried
one mathematically intensive technique to analyze the microcrystals. It
showed a fuzzy picture similar to other fibrils, telling them they were
on the right track but not giving them the details they were
seeking.
Nelson picked up the project four years ago. "Because the crystals
were so small, we ended up trying lots of techniques," she said. "We
formed collaborations with people who were experts in those areas.
Every time we'd come up with a new idea, it was exciting. Then, when we
were able to determine the structure, it was twice as exciting."
The breakthrough came when the researchers teamed up with European
crystallographer Christian Riekel, who had designed and built a special
x-ray beam as narrow as the crystals were tiny, and Anders Madsen, a
Danish student working at the synchrotron in France who was skilled in
special methods for mounting and manipulating the samples to collect
good x-ray diffraction data.
"With the first calculation, we were able to see the structure and
how we would be able to model atoms into that map," Nelson said. "That
was really the ah-ha moment."
The final detailed structure is broadly consistent with other
lower-resolution models, such as the two stacked beta sheets composed
of the main chain of amino acids. The surprise came with the molecular
side chains that give each amino acid its unique identity and hold the
pairs of beta sheets in formation.
Nelson expected to see only the ends of the side chains reaching out
and touching each other, they way they do in the DNA double helix.
Instead, she found interdigitated connections akin to zippers and
Velcro.
"This gives a structural explanation about why the fibers grow
almost infinitely, and why prions are infectious," said Roland Riek of
The Salk Institute. "Like a zipper, you have one end that never ends;
you always have a free binding site for a growing fiber." In a related
paper in Nature, Riek reported that the infectious ability of a
fungal prion depends on its beta sheet structure, which he proposed
would look similar to the detailed structure from Eisenberg's lab.
In another interesting finding, the zipped up fibril core is dry.
"Proteins love water," said Eisenberg. "When they are soluble, there is
water all around them. When the zipper is formed, water is forced out
of the interface between the two beta sheets. Once you have this dry
interface, it's hard to open up. Imagine trying to pry open two long
pieces of Velcro."
In preliminary follow-up experiments, Eisenberg and his colleagues
have found 10 short segments from other amyloid and prion proteins from
hamsters, mice, and people that exhibit the same behavior in the test
tube.
"We think virtually any protein can be converted into this type of
structure," said Christopher Dobson of Cambridge University, United
Kingdom, who wrote an accompanying commentary in Nature. "This
is the first model that gives an atomic-level image of how the
molecules might be stacked together in such a fibril."
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Jennifer Michalowski
