BERKELEY, CA --
A 30-year quest to solve the structure of one of the most important types of proteins in a living cell has been achieved. Scientists with the U.S. Department of Energy's Lawrence Berkeley National Laboratory have created the first 3-dimensional atomic model of tubulin, a protein that makes possible such vital life processes as cell division and the movement of materials within cells.
Eva Nogales, Sharon Wolf, and Kenneth Downing, biophysicists with
Berkeley Lab's Life Sciences Division, announced the completion of their model
in the January 8, 1998 issue of the scientific journal
Nature. At a resolution
of 3.7 angstroms, the model provides the first highly-detailed
three-dimensional look at tubulin, including the site where the protein
interacts with the promising anti-cancer drug taxol.
A ribbon diagram based on the 3.7 angstrom resolution model of tubulin developed by Berkeley Lab scientists shows the protein to be a dimer consisting of two monomers that are almost identical in structure. Each monomer is formed by a core of two beta sheets (blue and green) surrounded by helices, and each binds to a guanine nucleotide (pink). In addition to a nucleotide binding site, each monomer also has two other binding sites, one for proteins, and one for the anti-cancer drug taxol. |
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Tubulin is the protein that polymerizes into long chains or filaments that
form microtubules, hollow fibers which serve as a skeletal system for living
cells. Microtubules have the ability to shift through various formations which
is what enables a cell to undergo mitosis or to regulate intracellular
transport. The formation-shifting of microtubules is made possible by the
flexibility of tubulin which is why scientists have sought to understand the
protein's atomic structure since its discovery in the 1950s.
Interest in tubulin structure heated up intensely in recent years when
taxol, a natural substance found in the bark of the Pacific yew tree (the name
"taxol" has been trademarked by Bristol-Myers-Squibb), was shown in clinical
tests to be an effective treatment for a number of cancers including ovarian,
breast, and lung. Cancer occurs when cell division runs amok.
By binding to tubulin and causing the protein to lose its flexibility,
taxol prevents a cell from dividing. With better knowledge of tubulin
structure and its interaction with taxol, scientists believe that an even more
effective anti-cancer drug, one that interacts only with the tubulin of
cancerous cells, could be synthesized.
"An awful lot of people have wanted to know what tubulin looks like at the
atomic level," says Nogales. "The knowledge gained from this model should be
of invaluable help in understanding the microtubule system in the cell."
Tubulin is a "heterodimer" protein, meaning it is comprised of a pair of
polypeptide chains -- called monomers -- that differ in the sequence of their
amino acids. The model presented by Nogales, Wolf, and Downing, shows that
each tubulin monomer -- the alpha and the beta -- is a compact molecular
structure with three functional components or domains, one that binds to
nucleotides, one that binds to drugs like taxol, and one that looks to be a
binding site for other proteins.
"The interaction between domains is very tight so that the effects that
nucleotides, drugs, and other proteins have on tubulin are firmly linked," says
Nogales. "The assembly of tubulin and its regulation through dynamic
instability results from the fine tuning of the three components."
To produce their model, the Berkeley Lab researchers first polymerized
tubulin proteins under the same conditions in which microtubules are formed
except for the additional presence of zinc. The zinc prevents tubulin chains
from curling around into hollow fibers. Instead, the polymerized tubulin forms
two-dimensional crystalline sheets that are ideal for imaging by electron
crystallography. The use of electron-based rather than the x-ray-based
crystallography techniques customarily employed in protein studies was crucial
to the model's 3.7 angstrom resolution.
"Working with x-ray crystallography requires much more protein than
electron crystallography," says Downing. "Obtaining diffraction patterns with
an electron beam enables us to work with crystals only one molecule in
thickness, giving us our high resolution."
The final 3-D model is a computerized reconstruction derived from a data set that included 93 electron diffraction patterns and
159 images culled by the researchers from the more than 4,000 images which were
recorded over a six-year period. The diffraction patterns and images were
generated on an electron microscope equipped with a special "cold stage" that
reduced damage to the crystals from the electron beam and yielded less "noise"
than a conventional electron microscope. This microscope is also equipped to
allow the tilting of samples at various angles so images can be obtained from
"We needed to keep our samples extremely stable during the imaging process
in order to get the resolution we wanted," says Nogales. To this end, she and
her colleagues used taxol to lock their samples into fixed positions for
imaging. That the taxol would bind to their crystals is in part a confirmation
that their structural model of tubulin is accurate.
The Berkeley Lab is a U.S. Department of Energy national laboratory
located in Berkeley, California. It conducts unclassified scientific research
and is managed by the University of California.