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News and Publications
Macromolecular Assemblies Visualized by Electron Cryo-Microscopy and Image Processing: Membrane Proteins and Viruses
M. Yeager, B. Adair, S. Bacon, L. Brill, A. Cheng, L. Craig, M.J. Daniels, K.A. Dryden, L. Hsu, S. Lewis, T. Macke, R. Nunn, D. Schweissinger, M. Tihova, R.N. Beachy,* A.R. Bellamy,** J.A. Berriman,*** M. Buchmeier,** K. Coombs,**** C. Fauquet,* H.B. Greenberg,***** J.E. Johnson,+ N. Kumar,+ S. Matsui,***** A. Olson,+ L.H. Philipson,++ A. Rein,+++ A. Schneeman,+ 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
**** University of Manitoba, Winnipeg, Manitoba
***** Stanford University, Stanford, CA
+ TSRI
++ 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, my colleagues and I use high-resolution electron cryo-microscopy and computer image processing. With this approach, we can examine the molecular architecture of supramolecular assemblies such as membrane proteins and viruses.
In electron cryo-microscopy, 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 structures that can be related to the functional properties of such assemblies.
Research projects under way include the structural analysis of (1) membrane proteins involved with cell-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 (reovirus, nodavirus, tetravirus, and sobemovirus). 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 that cause sudden death.
We expressed a recombinant cardiac gap junction protein, a1-connexin, and produced 2-dimensional crystals suitable for electron cryo-crystallography. The 3-dimensional map (Fig. 1) shows that 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.
RNA VIRUSES
RNA viruses are the largest and most diverse group of pathogenic organisms. Of the estimated 17 million deaths per year caused by all infectious pathogens, at least two thirds can be attributed to these types of viruses. We discovered a precursor procapsid particle of nudaurelia capensis <OMEGA> virus (N<OMEGA>V), a small, nonenveloped RNA virus, that differs strikingly from the mature virion (Fig. 2).
The coordinates of the N<OMEGA>V protein subunit, which were previously determined by crystallography, were modeled into the 28-Å-resolution electron cryo-microscopy map of the procapsid. The resulting pseudoatomic model of the N<OMEGA>V procapsid showed the large rearrangements in quaternary and tertiary structure needed for the maturation of viruslike particles and presumably for maturation of the virus.
On the basis of this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change. These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses.
PUBLICATIONS
Brill, L.M., Nunn, R.S., Kahn, T.W., Yeager, M., Beachy, R.N. Recombinant tobacco mosaic virus movement protein is an RNA-binding, a-helical membrane protein. Proc. Natl. Acad. Sci. U. S. A. 97:7112, 2000.
Nunn, R.S., Macke, T.J., Olson, A.J., Yeager, M. Transmembrane a-helices in the gap junction membrane channel: Systematic search of packing models based on the pair potential function. Microsc. Res. Tech. 52:344, 2001.
Opalka, N., Tihova, M., Brugidou, C., Kumar, A., Beachy, R.N., Fauquet, C.M., Yeager, M. Structure of native and swollen sobemoviruses by electron cryo-microscopy and image reconstruction. J. Mol. Biol. 303:197, 2000.
Qu, C., Liljas, L., Opalka, N., Brugidou, C., Yeager, M., Beachy, R.N., Johnson, J.E., Lin, T. 3D domain swapping modulates the stability of members of an icosahedral virus group. Struct. Fold. Des. 8:1095, 2000.
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, in press.
Tang, L., Johnson, K.N., Ball, L.A., Lin, T., Yeager, M., Johnson, J.E. The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA. Nat. Struct. Biol. 8:77, 2001.
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., in press.
Yeager, M., Unger, V.M. Culturing of mammalian cells expressing recombinant connexins and two-dimensional crystallization of the isolated gap junctions. Methods Mol. Biol. 154:77, 2001.
Molecular Basis of Restenosis After Coronary Artery Angioplasty and Stent Placement
M. Yeager, E. Kaback,* J.C. Apostol,** P.D. Silva,** L. Wodicka,*** R.J. Russo*
* Department of Cell Biology, TSRI
** Scripps Clinic, La Jolla, CA
*** Genomics Institute of the Novartis Research Foundation, San Diego, 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 atherosclerosis. A recent advancement in the treatment of coronary atherosclerosis is percutaneous transluminal coronary angioplasty combined with implantation of a balloon-expandable stent that acts as a metallic scaffold to maintain patency of the diseased vessel. An adverse consequence of this procedure, which usually occurs within 3 to 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.
The identification of 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.
During the past 2 years, several milestones were accomplished in this research program: (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 vessels with 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-receptor 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.
Yeager Website
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