Friday, June 12,
2009
New research by scientists at The
Scripps Research Institute and other institutions provides a
close-up look at the cone-shaped shell that is the hallmark of
human immunodeficiency virus (HIV), revealing how it is held
together—and possible ways to break it apart.
Previously, scientists had known that the genetic material within
HIV is enclosed within a shell called the capsid, which is formed
by a honeycomb arrangement of about 250 hexagonal protein building
blocks. For HIV to infect human cells, the virus binds to cell
surface receptors, and then the capsid is delivered into the
cytoplasm of the cell.
Now, in an advance, online issue of the journal Cell published on
June 11, 2009, Professor Mark Yeager and colleagues at The Scripps
Research Institute, the University of Virginia, and the University
of Utah describethe first high-resolution molecular structure of
the hexagonal protein building block, called CA, that makes up the
HIV capsid. This detailed description may help scientists identify
new ways to block HIV infection.
Bringing Down the Capsid
Since HIV/AIDS was first recognized in 1981, several drugs and drug
combinations have allowed infected individuals to live longer and
healthier lives. However, resistance to the existing drugs has
created an urgent need for novel therapeutic strategies.
Current drugs target critical steps in the virus life cycle. For
example, protease inhibitors block the protein cleavages that
generate viral components—one of them being the protein CA.
Other possible ways to block infection would be to prevent
formation of the capsid by blocking assembly of CA molecules or to
find a way to disassemble the capsid once it is made.
"Anything that destabilizes the capsid, either by inhibiting
assembly or accelerating disassembly should attenuate or even kill
the virus," says Owen Pornillos, an investigator in Yeager's lab
and first author of the Cell paper.
But to destabilize the capsid, it's necessary to know precisely how
it is held together.
Making Crystals
"No one had been able to visualize the CA hexamer at atomic
resolution," says Yeager. "Other groups had been able to solve
structures of individual regions of CA. But it was not clear from
these structures exactly how the CA proteins fit together."
To make the capsid, sets of six CA protein molecules first form
hexamers, which then associate with one another to build a
honeycomb-like shell comprised of about 250 hexamers. The ends of
the shell are closed by insertion of seven and five CA protein
pentamers, yielding the characteristic cone-like appearance of the
capsid.
In 2007, Yeager's group managed to view the CA hexamers by a type
of electron microscopy in which the samples are quick frozen in
buffers, which preserves the inherent structure of proteins. That
study provided the first glimpse of how CA proteins are arranged in
the capsid. (The first author of the 2007 article was Barbie
Ganser-Pornillos, Owen Pornillos' wife, who was also involved in
the current study.)
In order to view the CA hexamer at even higher resolution, Yeager's
group turned to X-ray crystallography. This technique requires
growing 3D crystals of a molecule and then scattering a beam of
X-rays off the crystals, which are recorded on a detector.
Computational methods are then used to interpret the scattering
patterns to calculate the position of every atom in the
crystallized molecule.
But growing large, 3D crystals of the CA hexamer was no easy feat.
The two ends of each CA protein molecule are held together by a
"floppy" bridge, which precluded formation of orderly arrays of CA
hexamers to form 3D crystals.
To overcome the problem, Pornillos and Yeager turned to molecular
biology. They engineered CA proteins that would form sturdy
chemical links between them, relying on the 2007 structure as their
roadmap to determine exactly where to place the links.
"Our work takes advantage of so-called hybrid methods—molecular
biology, biochemistry, electron microscopy, and X-ray
crystallography," says Yeager. "These methods are synergistic. The
EM results guided the molecular biology to engineer stable CA
hexamers that were then amenable to 3D crystallization and X-ray
structure analysis at atomic resolution."
The structure they obtained provided a view of the CA hexamer at an
unprecedented resolution of two-Ångstrom (one Ångstrom equals one
ten-billionth of a meter).
A Close-Up Look
All proteins are composed of linear chains of amino acids—with one
end called the N-terminus and the opposite end the C-terminus—that
are folded in three-dimensional shapes. In the CA protein, amino
acid chains are twisted into several rods, called a-helices, with
extensions—called side chains—that protrude from the main chain to
interact with other folded regions of the protein.
The two-Ångstrom structure showed the positioning of these
a-helices and, for the first time, the location of the atoms in the
side chains. "We could precisely delineate all the chemical
interactions that stabilize the hexamer," says Yeager.
The center of the CA hexamer is formed by 6 N-terminal ends of the
CA protein subunits. The C-terminal domains form a "floppy" belt
around this central core, connecting adjacent hexamers. The fact
that the belt is not held rigidly in place, helps explain how the
honeycomb shape of the capsid forms. "The curvature of the capsid
is not constant," says Pornillos. "Now we can see in atomic detail
how flexibility in CA makes this happen."
The group discovered another set of interactions critical to
stabilizing the capsid—connections between the N-terminal and
C-terminal ends of adjacent CA protein molecules in one hexamer.
"Think of the fingers of one hand as the N-terminal domain and the
palm as the C-terminal," says Yeager. "Imagine the fingers of one
hand being cradled in the palm of the other, and so on as if you
had six hands in a ring."
Knowing precisely how and where CA proteins interact gives
researchers clues on how to interfere with these connections. One
approach is to design small molecules that can insert themselves at
strategic positions, impeding capsid assembly or making the capsid
less stable.
While finding HIV therapies is a main driver for Yeager's work, he
points out that it also provides fundamental insights into biology.
"Determining the assembly of a relatively simple structure like the
capsid of a virus can help us understand how more complex
biological structures inside the cell are organized," he
explains.
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Scripps Research Institute
Thanks to Scripps Research
Institute for this article.