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News and Publications
Macromolecular Assemblies Visualized by Electron Cryomicroscopy
and Image Processing: Membrane Proteins and Viruses
M. Yeager, B. Adair, S. Bacon, L. Brill, A. Cheng, L. Craig,
M.J. Daniels, F. Dawood, K.A. Dryden, M. Tihova, R.N. Beachy,* A.R.
Bellamy,** J.A. Berriman,*** M. Buchmeier,**** K. Coombs,***** C.
Fauquet,* H.B. Greenberg,+ J.E. Johnson,**** S. Matsui,+
A. Olson,**** L.H. Philipson,++ A. Rein,+++
A. Schneemann,**** J.A. Tainer,**** J.A. Taylor**
*Donald Danforth Plant Science Center, St. Louis, MO
** University of Auckland, Auckland, New Zealand
*** MRC Laboratory of Molecular Biology, Cambridge, England
**** TSRI
***** University of Manitoba, Winnipeg, Manitoba
+ Stanford University, Stanford, CA
++ University of Chicago, Chicago, IL
+++ Frederick Cancer Research Facility, Frederick, MD
We are intrigued by biological questions at the interface between
cell biology and structural biology: How do membrane proteins fold?
How do membrane channels open and close? How are signals transmitted
across a cellular membrane when an extracellular ligand binds to
a membrane receptor? How do viruses attach and enter host cells,
replicate, and assemble infectious particles? To explore such problems,
we use high-resolution electron cryomicroscopy and computer image
processing. With this approach, we can examine the molecular architecture
of supramolecular assemblies such as membrane proteins and viruses.
In electron cryomicroscopy, biological specimens are quick frozen
in a physiologic state to preserve their native structure and functional
properties. A special advantage of this method is that we can capture
dynamic states of functioning macromolecular assemblies, such as
open and closed states of membrane channels and viruses actively
transcribing RNA. Three-dimensional density maps are obtained by
digital image processing of the high-resolution electron micrographs.
The rich detail in the density maps indicates the power of this
approach to reveal the structural organization of complex biological
systems that can be related to the functional properties of such
assemblies.
Research projects under way include the structure analysis of (1)
membrane proteins involved in cell-to-cell communication (gap junctions),
water transport (aquaporins), ionic transport (potassium channels),
transmembrane signaling (integrins), and viral recognition (rotavirus
NSP4); (2) viruses responsible for human diseases (rotavirus, astrovirus,
retroviruses); and (3) viruses used as model systems to understand
mechanisms of pathogenesis (reoviruses, nodaviruses, tetraviruses,
and sobemoviruses). The following sections summarize selected projects
that exemplify the themes of our research program.
CARDIAC GAP JUNCTION MEMBRANE CHANNELS
Cardiac gap junctions play an important functional role in the
heart by electrically coupling adjacent cells, thereby organizing
the pattern of current flow to allow a coordinated contraction of
the muscle. They are therefore intimately involved in both normal
coordinated depolarization of heart muscle and cardiac arrhythmias
causing sudden death.
We expressed a recombinant cardiac gap junction protein, a1-connexin, and produced
2-dimensional crystals suitable for electron cryocrystallography.
The 3-dimensional map (Fig. 1) shows that the dodecameric channel
is about 150 Å long, with a diameter of about 65 Å within
the membranes and about 55 Å in the extracellular gap. Within
the membrane interior, each hexameric connexon is formed by 24 rods
of density, consistent with an a-helical conformation for the 4 transmembrane domains of
each subunit. We anticipate that this basic molecular design will
be a common folding motif for gap junction channels.
ROTAVIRUS
Rotavirus causes severe gastroenteritis in infants and young animals
and is responsible for the death of approximately 700,000 children
per year in developing countries. The surface of rotavirus is decorated
with 60 spikelike projections, each composed of a dimer of VP4,
the viral hemagglutinin. Trypsin cleavage of VP4 generates 2 fragments:
VP8*, which binds sialic acid, and VP5*, which contains an integrin
binding motif and a hydrophobic region that permeabilizes membranes
and is homologous to fusion domains. Although the mechanism for
cell entry by this nonenveloped virus is unclear, it is known that
trypsin cleavage enhances viral infectivity and facilitates viral
entry.
We used electron cryomicroscopy and difference map analysis to
localize the binding sites for 2 neutralizing monoclonal antibodies,
7A12 and 2G4, which are directed against the sialic acid binding
site within VP8* and the membrane permeabilization domain within
VP5*, respectively (Fig. 2). 
The 7A12 antibody binds at the tips of the dimeric heads of VP4,
and 2G4 binds in the cleft between the 2 heads of the spike. When
these binding results are combined with secondary structure analysis,
we predict that the VP4 heads are composed primarily of b-sheets in VP8* and that VP5*
forms the body and base primarily in b-structure and a-helical
conformations, respectively. On the basis of these results and those
of others, a model is proposed for cell entry in which VP8* and
VP5* mediate receptor binding and membrane permeabilization, and
uncoating occurs during transfer across the lipid bilayer, thereby
generating the transcriptionally active particle.
PUBLICATIONS
Brugidou, C., Opalka, N., Yeager, M., Beachy, R.N., Fauquet,
C. Stability of rice yellow mottle virus and cellular compartmentalization
during the infection process in Oryza sativa (L). Virology
297:98, 2002.
Saffitz, J.E., Yeager, M. Intracardiac cell communication
and gap junctions. In: Foundations of Cardiac Arrhythmias.
Spooner, P.M., Rosen, M.R. (Eds.). Marcel Dekker, New York, 2001,
p. 171.
Tang, L., Lin, C.S., Krishna, N.K., Yeager, M., Schneemann,
A., Johnson, J.E. Virus-like particles of a fish nodavirus display
a capsid subunit domain organization different from that of insect
nodaviruses. J. Virol. 76:6370, 2002.
Tihova, M., Dryden, K.A., Bellamy, A.R., Greenberg, H.B., Yeager,
M. Localization of membrane permeabilization and receptor binding
sites on the VP4 hemagglutinin of rotavirus: implications for cell
entry. J. Mol. Biol. 314:985, 2001.
Restenosis After Coronary Artery Angioplasty and Stent Placement
M. Yeager, E. Kaback,* J.C. Apostol,** P.D. Silva,** J. Stroebel,*
R.J. Russo**
* Department of Cell Biology, TSRI
** Scripps Clinic, La Jolla, CA
Cardiovascular disease is the major cause of mortality in the
United States; most of these deaths are due to myocardial infarction
caused by coronary artery atherosclerosis. A recent advancement
in the treatment of coronary atherosclerosis is percutaneous transluminal
coronary angioplasty combined with implantation of a balloon-expandable
stent, which acts as a metallic scaffold to maintain patency of
the diseased vessel. An adverse consequence of this procedure, which
usually occurs within 3-12 months, is a proliferation of cells in
the wall of the artery, a process termed neointimal hyperplasia.
In many patients, neointimal hyperplasia narrows the lumen of the
vessel (i.e., causes restenosis) and results in impaired myocardial
blood flow.
Identifying specific inhibitors of neointimal hyperplasia that
could decrease the prevalence of restenosis after placement of a
coronary artery stent would have extraordinary clinical value. The
porcine in vivo coronary artery injury model most closely resembles
the process of restenosis after stent placement in humans and therefore
provides the best system for delineating the pathophysiology of
neointimal hyperplasia.
The availability of the human genome sequence and the development
of oligonucleotide microarray technology provide unprecedented opportunities
to understand and treat human disease. The pattern of mRNA abundance
could be a powerful diagnostic tool by providing a "molecular fingerprint"
that would be much more reliable than qualitative pathologic evaluation
of tissue sections. In addition, the pattern of gene expression
can be used to gain insight into the "molecular circuitry" of disease.
We are using this technology to explore the molecular basis of restenosis.
Thus far, we have accomplished the following milestones: (1) We
can routinely place coronary stents in pigs (51 arteries in 28 animals),
and our operative mortality is less than published values. (2) Porcine
coronary arteries can be rapidly harvested, and methods have been
optimized to minimize RNA degradation. (3) We showed that microarrays
of human oligonucleotides can be used to examine gene expression
in porcine tissue. (4) Expression data are available from 6 samples
of neointimal hyperplasia and 6 samples of untreated, control vessels.
Our preliminary analysis suggests that levels of mRNA for fibronectin,
cadherin, c-fos and phosphatidic acid phosphatase in vessels with
neointimal hyperplasia are dramatically changed. These results suggest
that signaling pathways involving extracellular matrix proteins
and protooncogenes participate in neointimal hyperplasia. Identification
of cell receptors and signaling pathways associated with stent-induced
vascular injury in this porcine model may guide the design of novel
treatments to prevent restenosis in humans.
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