News and Publications

Macromolecular Assemblies Visualized by Electron Cryomicroscopy and Image Processing: Membrane Proteins and Viruses

* Donald Danforth Plant Science Center, St. Louis, Missouri
** University of Auckland, Auckland, New Zealand
*** MRC Laboratory of Molecular Biology, Cambridge, England
**** TSRI
***** University of Manitoba, Winnipeg, Manitoba
⁺ Stanford University, Stanford, California
⁺⁺ University of Chicago, Chicago, Illinois
⁺⁺⁺ National Cancer Institute, Frederick, Maryland

The ultimate goal of our studies is to gain a deeper understanding of the molecular basis for important human conditions, such as sudden death, heart attacks, and HIV disease, that cause substantial mortality and suffering. The structural details revealed by our research may provide clues for the design of more effective and safer medicines.

At the basic science level, we are intrigued by 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 significant 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, α₁-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 diameters of about 65 Å within the membrane 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 α-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.

Integrins

Cardiovascular disease is the major cause of mortality in the United States; most of these deaths are due to myocardial infarction (heart attack) caused by coronary atherosclerosis. Myocardial infarction almost always is due to formation of a thrombus at the site of a coronary artery stenosis. A key event that stimulates thrombus formation is platelet aggregation, which is mediated by the prototypical integrin α_IIbß₃. Integrins are a large family of heterodimeric transmembrane receptor proteins that modulate cell adhesion, such as platelet aggregation, as well as other important biological processes, such as development, angiogenesis, wound healing, and neoplastic transformation. Integrins accomplish these diverse functions by mediating dynamic linkages between extracellular adhesion molecules and the intracellular environment. Integrin functions are regulated by transmembrane signaling, which can occur as a consequence of both binding of extracellular ligands (so-called "outside-in" signaling) and binding of molecules to the cytoplasmic domains (so-called "inside-out" signaling).

We used electron cryomicroscopy and single-particle image reconstruction to derive a 3-dimensional structure at 20-Å resolution of the unliganded, low-affinity state of the human platelet integrin α_IIbß₃. The large ectodomain and small cytoplasmic domains are connected by a rod of density that we interpret as 2 parallel transmembrane α-helices (Fig. 2). The docking of the x-ray structure of the α_vß₃ ectodomain into the electron cryomicroscopy map of α_IIbß₃ requires hinge movements at linker regions between domains in the crystal structure. Comparison of the putative high- and low-affinity conformations revealed dramatic conformational changes associated with integrin activation. The structural details revealed by these studies will provide insight into the molecular basis of integrin activation that will be relevant for the rational design of drugs to modulate integrin functions.

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 antibodies, 7A12 and 2G4, which are directed against the sialic acid-binding site within VP8* and the membrane permeabilization domain within VP5*, respectively (Fig. 3). 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 ß-sheets in VP8* and that VP5* forms the body and base primarily in ß-structure and α-helical conformations, respectively. On the basis of these results and those of others, we propose a model 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

Adair, B.D., Yeager, M. Three-dimensional model of the human platelet integrin α_IIbß₃ based on electron cryomicroscopy and x-ray crystallography. Proc. Natl. Acad. Sci. U. S. A. 99:14059, 2002.

Craig, L., Taylor, R.K., Pique, M.E., Adair, B.D., Arvai, A.S., Singh, M., Lloyd, S.J., Shin, D.S., Getzoff, E.D., Yeager, M., Forest, K.T., Tainer, J.A. Type IV pilin structure and assembly: x-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin. Mol. Cell 11:1139, 2003.

Ganser, B.K., Cheng, A., Sundquist, W.I., Yeager, M. Three-dimensional structure of the M-MuLV CA protein on a lipid monolayer: a general model for retroviral capsid assembly. EMBO J. 22:2886, 2003.

Restenosis After Coronary Artery Angioplasty and Stent Placement

* Scripps Clinic, La Jolla, California

Cardiovascular disease is the major cause of mortality in the United States. Most of these deaths are due to myocardial infarction (heart attack) 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. 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.

Oligonucleotide microarray technology provides unprecedented opportunities to understand and treat human disease. The pattern of mRNA abundance can be used to gain insight into the "molecular circuitry" of disease. We are using this technology to explore the molecular basis of restenosis. Our preliminary analysis suggests that levels of mRNA for several genes are dramatically changed. 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.